Figure 5. Muscle contraction and phosphorylation of MYPT1 and MLC₂₀ in response to PAR2-AP.

(A) Time course of PAR2-AP-induced contraction in dispersed gastric smooth muscle cells. PAR2-AP (1 µM) was added to freshly isolated muscle cells for different periods of time ranging from 30 s to 10 min. (B) Concentration-response curves for the contractile effect of PAR2-AP. PAR2-AP was added to freshly isolated muscle cells at a concentration ranging from 10 pM to 1 µM, and peak contraction at 30 s (solid circles) and sustained contraction at 10 min (solid triangles) were measured. (C) Phosphorylation of MYPT1, and MLC₂₀ by PAR2-AP in dispersed muscle cells. Freshly dispersed muscle cells were treated with PAR2-AP for 30 s and 10 min. MLC₂₀ phosphorylation was measured using phospho-specific-Ser¹⁹ MLC₂₀ antibody. MYPT1 phosphorylation was measured using phospho-specific-Thr⁶⁹⁶ MYPT1 antibody. (D and E) Effect of inhibitors on initial and sustained contraction in response to PAR2-AP. Freshly dispersed muscle cells were treated separately with the PLC inhibitor U73122 (10 µM), MLC kinase inhibitor ML-9 (10 µM), PKC inhibitor bisindolylmaleimide (1 µM), Rho kinase inhibitor Y27632 (1 µM) for 10 min, or pertussis toxin (200 ng/ml) for 60 min, and then treated with PAR2-AP (1 µM) for 30 s (initial) or 10 min (sustained). Muscle contraction was measured by scanning micrometry, and the results are expressed as percent decrease in cell length from control cell length (105±3 µm). Values are means ± S.E. of six experiments. ** Significant inhibition from control response (P<0.001).