Fig. 5.

OA-induced inhibition of Rho kinase activity via Epac/Rap1. A: freshly dispersed gastric smooth muscle cells were treated with OA (10 μM) for 10 min in the presence or absence of myristoylated PKI (1 μM), and Rap1 activity was determined using GST-fusion protein containing the Rap1 binding domain of Rap1GDS to affinity precipitate active Rap1 (GTP-Rap1) as described in methods. Expression of Rap1 in cells transfected with control siRNA or Rap1 siRNA was analyzed by Western blot. B and C: cultured muscle cells transfected with control vector or vector containing Rap1 siRNA were treated with CCh (0.1 μM) in the presence or absence of OA (10 μM) or 8-pCPT-2′-O-Me-cAMP (10 μM) for 10 min. In some experiments cell were incubated with myristoylated PKI (1 μM). Rho kinase activity was measured by immunokinase assay as described in materials and methods. Results are expressed as cpm/mg protein. Values are means ± SE of 4 experiments. **P < 0.01 vs. basal. ##P < 0.01 vs. CCh.