Abstract
Myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) govern myosin light chain (LC20) phosphorylation and smooth muscle contraction. Rho kinase (ROK) inhibits MLCP, resulting in greater LC20 phosphorylation and force generation at a given [Ca(2+) ](i) . Here, we investigate the role of ROK in regulating LC20 phosphorylation and spontaneous contractions of gastric fundus, gastric antrum, and proximal colon smooth muscles. Protein and phosphorylation levels were determined by western blotting. The effects of Y27632, nicardipine, and GF109203X on phosphorylation levels and contraction were measured. γ-Actin expression is similar in all three smooth muscles. LC20 and pS19 are highest, but ROK1 and ROK2 are lowest, in antrum and proximal colon smooth muscles. LZ +/- myosin phosphatase targeting subunit 1 (MYPT1), CPI-17, and pT696, pT853, and pT38 are highest in fundus and proximal colon smooth muscles. Myosin phosphatase-rho interacting protein (M-RIP) expression is lowest in fundus, and highest in antrum and proximal colon smooth muscles. Y27632 reduced pT853 in each smooth muscle, but reduced pT696 only in fundus smooth muscles. Nicardipine had no effect on pT38 in each smooth muscle, while GF109203X reduced pT38 in proximal colon and fundus smooth muscles. Y27632 or nicardipine reduced pS19 in proximal colon and fundus smooth muscles. Y27632 or nicardipine inhibited antrum and proximal colon smooth muscle spontaneous contractions, but only Y27632 reduced fundus smooth muscle tone. Zero external Ca(2+) relaxed each smooth muscle and abolished LC20 phosphorylation. Organ-specific mechanisms involving the MLCP interacting proteins LZ +/- MYPT1, M-RIP, and CPI-17 are critical to regulating basal LC20 phosphorylation in gastrointestinal smooth muscles.
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