Fig. 5.

PDE inhibitors do not increase cGMP in colon cancer cell lines due to a lack of endogenous cGMP production. The expression of functional guanylyl-cyclase-C (GC-C) in colon cancer cells engineered to express ectopic PKG2 was determined by measuring VASP phosphorylation in response to treatment with the GC-C agonist linaclotide. (A) Different cell lines induced to express PKG2 as indicated below, were either untreated (control, C), treated with 8Br-cGMP (cG), or with the combination of linaclotide and sildenafil (L+S). Western blots were probed for β-actin as a loading control for PKG2 expression. (B) Dose-response study of linaclotide stimulation of LS174T-PKG2 cells to determine the dose required to generate minimal cGMP/PKG2 activity as measured by VASP phosphorylation. (C) LS174T-PKG2 cells were treated with PDE inhibitors as indicated, in the presence of 10 nM linaclotide. Relative cGMP levels were then measured by immunoblotting for VASP-239P. All immunoblots were reprobed with antitotal VASP to indicate relative phosphorylation.