Regulation of basal LC20 phosphorylation by MYPT1 and CPI-17 in murine gastric antrum, gastric fundus, and proximal colon smooth muscles

Abstract

Background: 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.

Methods: Protein and phosphorylation levels were determined by western blotting. The effects of Y27632, nicardipine, and GF109203X on phosphorylation levels and contraction were measured.

Key results: 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.

Conclusions & inferences: 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.

Figure 1

Expression levels of γ-actin, LC20, pS19, ROK1, ROK2, total MYPT-1, LZ+, LZ− MYPT1, M-RIP, pT696, pT853, CPI-17, and pT38 in antrum, proximal colon, and fundus smooth muscles. Upper panels, cumulative data of average density values (+/− SD) of each protein in each smooth muscle tissue. Lower panels, representative western blots. A. LC20, I. ROK1, ROK2, E. M-RIP * average density significantly different from antrum and proximal colon (P<0.01); antrum and proximal colon average densities not significantly different from each other. B. CPI-17, C. total MYPT1, D. LZ+, LZ− MYPT1, H. pT696, pT853 * average densities significantly different from antrum (P<0.01); proximal colon and fundus average densities not significantly different from each other. F. pS19, G. pT38 * average densities significantly different from antrum (P<0.01); proximal colon and fundus average densities significantly different from each other (P<0.01). J. γ-actin average densities not significantly different from each other. γ-actin, 10 μg protein/lane; M-RIP, 25 μg protein/lane; all other proteins, 20μg protein/lane. Antrum (A), proximal colon (PC), and fundus (F) smooth muscle homogenates from at least 7 mice were analyzed by densitometry of duplicate or triplicate western blots.

Figure 2

Effects of Y27632 and nicardipine on basal pT696, and pT853 in antrum, proximal colon, and fundus smooth muscles. Top panels, representative western blots, 20 μg protein/lane. Middle panels, pT696, cumulative data of average density values (+/− SD). Bottom panels, pT853, cumulative data of average density values (+/− SD). C, control; nic, nicardipine (1μM). * average density values significantly different from control values (P<0.05). At least 4 antrum, fundus, and proximal colon smooth muscle homogenates from each treatment were analyzed by densitometry of duplicate or triplicate western blots.

Figure 3

Effects of Y27632, GF109203X, and nicardipine on basal pT38 in antrum, proximal colon, and fundus smooth muscles. Upper panels, cumulative data of average density values (+/− SD) of pT38 in each smooth muscle tissue. Lower panels, representative western blots, 20 μg protein/lane. *P<0.05, ^#P<0.01, average density values significantly different from control values (no drugs present). At least 4 antrum, fundus, and proximal colon smooth muscle homogenates from each treatment were analyzed by densitometry of duplicate or triplicate western blots.

Figure 4

Effects of Y27632 and nicardipine on basal pS19 in antrum, proximal colon, and fundus smooth muscles. Upper panels, cumulative data of average density values (+/−SD) of pS19 in each smooth muscle tissue. Lower panels, representative western blots, 20 μg protein/lane. C, control; nic, nicardipine (1μM). * average density values significantly different from control values (P<0.05). At least 4 antrum, fundus, and proximal colon smooth muscle homogenates from each treatment were analyzed by densitometry of duplicate or triplicate western blots.

Figure 5

Effects of wortmannin or zero Ca²⁺ Kreb’s on basal pS19 in antrum, proximal colon, and fundus smooth muscles. std, 20kDa protein marker; C, control. At least 3 antrum, fundus, and proximal colon smooth muscle homogenates from each treatment were analyzed by densitometry of duplicate western blots.

Figure 6

Effects of Y27632 or nicardipine on basal contractions of antrum, proximal colon, and fundus smooth muscles. Representative traces of antrum (A), proximal colon (B), and fundus (C) smooth muscles, recorded as described in Materials and Methods. D. cumulative data from 5 antrum, proximal colon, and fundus smooth muscles of the average percent inhibition of basal contraction (+/− SD). The relaxation of each smooth muscle tissue by 1μmol L⁻¹ nicardipine and 10μmol L⁻¹ SNP together was taken as 100% percent inhibition. *P < 0.05, **P < 0.01, significantly different from the control regions preceding the drugs.