Proximity Ligation Assay of Interactions between β‐1 Integrin and Ca2+ Sensitization Proteins During the Contractile Response of Gastric Fundus Smooth Muscles to Cholinergic Stimuli

Abstract

Contraction and relaxation of smooth muscle involves the regulation of myosin light chain (LC20) phosphorylation by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Rho‐associated, coiled‐coil containing protein kinase 2 (ROCK2), and myosin phosphatase target subunit 1 (MYPT1) are crucial for gastrointestinal smooth muscle contractile regulation via their inhibition of MLCP activity. β‐1 integrin is a trans‐membrane receptor that forms focal contacts and mediates the attachment of smooth muscle cells to the extracellular matrix and therefore plays an important role in cell adhesion, tension generation, and mechanotransduction in smooth muscle cells.Here we use proximity ligation assays (PLA) to examine the role of β‐1 integrin in gastric fundus smooth muscle contraction. Muscle contraction with bath‐applied carbachol (CCh) or neurally‐released ACh by atropine‐sensitive electrical field stimulation (EFS) significantly increased the interaction of MYPT1, ROCK2 and S19 phosphorylated LC20 with β‐1 integrin. Furthermore, PLA analysis of intact gastric fundus smooth muscle tissue shows that both bath‐applied CCh and neurally‐released ACh increase MYPT1‐T853 phosphorylation, in contrast to our previous findings obtained by western blot analysis of whole cell lysates showing that only CCh treatment increased MYPT1‐T853 phosphorylation. These results indicate that Ca2+ sensitization and contractile proteins interact with β‐1 integrin in focal contacts in response to cholinergic stimuli, indicating a potentially important role of β‐1 integrin in contributing to the contractile responses of fundus smooth muscles to cholinergic stimuli.