PMC

Contractile activity measured 6 hours after surgery in the distal small intestine in EDEMA and CONTROL groups is shown as follows: Panel A. Average basal tone; Panel B. Average basal tone with changes in calcium concentrations; Panel C. Average cycle amplitude; Panel D. Average cycle amplitude with changes in calcium concentration. (n= 19 per group in Panels A and C; n= 4–5 per group in Panels B and D; pCa= −log[Ca²⁺]; *, p<0.05 vs. CONTROL; +, p<0.05 vs. pCa=5.5)

The role of Zipper-interacting kinase (ZIPK) in edema-induced suppression of intestinal smooth muscle MYPT1 phosphorylation is shown. Panel A. ZIPK activity towards MYPT1 peptide substrate, as measured in the in vitro phosphate incorporation assay, in intestinal smooth muscle tissue lysates from CONTROL and EDEMA groups collected 6 hours after surgery. Panel B. ZIPK protein levels normalized to GAPDH in intestinal smooth muscle from CONTROL and EDEMA groups 6 hours after surgery. Panel C. MYPT1 phosphorylation activity in intestinal smooth muscle tissue lysates from CONTROL animals after treatment with Y-27632, SM1 or a combination of both inhibitors. (Panel A: n=6 in CONTROL and n=8 in EDEMA; Panel B: n=6 per group; Panel C: n=6 per group; Panel A: *, p<0.05 vs. CONTROL; Panel C: *, p<0.05 vs. Total; +, p<0.05 vs. Y-27632)

Theoretical model of decreased intestinal smooth muscle myosin light chain phosphorylation after edema development. Edema induces decreased rhoA activation and thus less rho-associated protein kinase (ROCK) activity. Less ROCK activity results in less ZIPK activity and thus less phosphorylation of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase. Alternatively (shown in blue), edema may independently inhibit both ROCK and ZIPK which then result in decreased MYPT1 phosphorylation. The catalytic subunit (PP1) of myosin light chain phosphatase is normally constitutively inhibited. However, decreases in MYPT1 phosphorylation induced by edema leads to less inhibition of the phosphatase and, therefore, more phosphatase activity. According to the functional studies, myosin light chain kinase (MLCK) activity is decreased. More myosin light chain phosphatase activity and less myosin light chain kinase activity leads to less myosin light chain (MLC) phosphorylation and therefore, less contractile activity.

The effects of myosin light chain kinase activity on contractile activity are shown. Panels A and B. Samples tracing of spontaneous contractions in the distal small intestine 6 hours after surgery in CONTROL and EDEMA groups, respectively, before and after treatment with the myosin light chain kinase inhibitor, W-7 (50 uM). Panel C. Inhibition of contractile amplitude and basal tone after treatment with W-7 in CONTROL and EDEMA groups. Panel D. Changes in myosin light chain kinase activity over time measured in tissue lysates from CONTROL and EDEMA groups. (Panel C, n= 4 per group; Panel D, n= 5 per group in 0–2 hour time points, n= 10 per group at 6 hour time point; *, p<0.05 vs. CONTROL)

The effects of edema on MYPT1 phosphorylation are shown. Panel A. Change in contractile activity from baseline after inhibition of the catalytic subunit of myosin light chain phosphatase with okadaic acid to (1 nM). Panel B. Phosphorylation of an MYPT1 peptide substrate measured in vitro after treatment with intestinal smooth muscle tissue lysates from CONTROL and EDEMA groups collected at 0, 0.5, 2 and 6 hour time points. Panel C. Representative Western blots showing phosphorylation of MYPT1 at thr696 and Thr853. Panel D. The ratio of phosphorylated to unphosphorylated MYPT1 in intestinal smooth muscle from CONTROL and EDEMA groups 6 hours after surgery. Panel E. The ratio of total MYPT1 (including phosphorylated and unphosphorylated MYPT1) to GAPDH in intestinal smooth muscle from CONTROL and EDEMA groups 6 hours after surgery. (Panel A, n=4 per group; Panel B, n=5–6 per group for 0–2 hour time points, n=9 per group for 6 hour time point; Panel D, n=11 per group for Thr(696), n=9 per group for Thr(853), Panel E, n=4 per group; *, p<0.05 vs. CONTROL; +, p<0.05 vs. 0 time point)

The role of rho kinase (ROCK) in edema-induced suppression of intestinal contractile activity is demonstrated. Panel A. Inhibition of contractile activity after treatment with rho kinase inhibitor (Y-27632, 10 uM) alone, Y-27632 treatment after pretreatment with a myosin light chain kinase inhibitor (W-7, 50 uM), and Y-27632 treatment after pretreatment with a myosin light chain phosphatase inhibitor (Okadaic acid, 250 nM). Panel B. ROCK activity towards an MYPT1 peptide substrate in intestinal smooth muscle lysates from CONTROL and EDEMA groups collected 6 hours after surgery. Panel C. rhoA GTPase activity in intestinal smooth muscle tissue lysates from CONTROL and EDEMA groups measured as rhoA-GTP binding to an immobilized effector molecule. Panel D. Representative Western blot of ROCK2 in CONTROL and EDEMA groups 6 hours after surgery. Panel E. Quantification of ROCK2 protein levels normalized to GAPDH in intestinal smooth muscle from CONTROL and EDEMA group at 6 hour time point (GAPDH, glyceraldehydes dehydrogenase). Panel F. Representative Western blot of rhoA in CONTROL and EDEMA groups 6 hours after surgery. Panel G. Quantification of rhoA protein levels normalized to GAPDH in intestinal smooth muscle from CONTROL and EDEMA group at 6 hour time point. (Panel A, n=5 per group for no pretreatment and okadaic acid pretreatment group, n=3 for W-7 pretreatment groups; Panel B, n=10 per group; Panel C, n=5 per group; Panel E, n=9 per group; Panel G, n=9 per group; *, p<0.05 vs. CONTROL)