Activation of Guanosine 3′,5′-Cyclic Monophosphate (cGMP)-Dependent Protein Kinase in Rat Vas Deferens and Distal Colon is Not Accompanied by Inhibition of Contraction1

Abstract

There is good evidence that in vascular smooth muscle, the relaxant effects of sodium nitroprusside (SNP) are mediated by increases in cGMP levels and activation of cGMP-dependent protein kinase (PKG). However, in rat vas deferens and rat distal colon, cGMP-elevating agents such as SNP and atrial natriuretic factor (ANF) have been shown to elevate cGMP without inducing relaxation. The lack of relaxation might be explained by either lack of activation of PKG by these agents or low levels of PKG in these tissues. The object of the present study was to investigate these possibilities by simultaneously monitoring cGMP levels, PKG activity and contractility in isolated strips of rat vas deferens, rat proximal colon and distal colon exposed to high concentrations of SNP or ANF. Verification of the specificity of the assay for PKG was obtained using MonoQ chromatography to resolve soluble smooth muscle extracts, followed by immunoblotting with a PKG-specific antibody to identify the kinase. In rat vas deferens, 5 mM SNP increased cGMP levels (14-fold) and PKG activity ratios (3.4-fold) but did not inhibit phenylephrine-induced contractions. In both rat proximal and rat distal colon, 100 nM ANF significantly elevated cGMP levels and PKG activity ratios, but only in the proximal colon was inhibition of spontaneous contractions observed. Total PKG activity was much lower (≈16 pmol PO₄/min/mg protein) in rat vas deferens, which was not relaxed by SNP, than in rabbit aorta (≈148 pmol PO₄/min/mg), which was relaxed. However, in the rat proximal colon, despite low PKG levels (≈11 pmole/min/mg), ANF did inhibit contractions. Thus the inability of the cGMP-elevating agents SNP and ANF to inhibit contractions in rat vas deferens and rat distal colon cannot be explained by either of the possibilities suggested above.