KT5823 inhibits cGMP-dependent protein kinase activity in vitro but not inintact human platelets and rat mesangial cells

Many signal transduction pathways are mediated by the second messengers cGM P and cAMP, cGMP- and cAMP-dependent protein kinases (cGK and PHA), phospho diesterases, and ion channels. To distinguish among the different cGMP effe cters, inhibitors of cGK and PKA have been developed including the K-252 co mpound KT5823 and the isoquinolinesulfonamide H89, KT5823, an in vitro inhi bitor of cGK, has also been used in numerous studies with intact cells to i mplicate or rule out the involvement of this protein kinase in a given cell ular response, However, the efficacy and specificity of KT5823 as cGK inhib itor in intact cells or tissues have never been demonstrated. Here, we anal yzed the effects of both KT5823 and H89 on cyclic-nucleotide-mediated phosp horylation of vasodilator-stimulated phosphoprotein (VASP) in intact human platelets and rat mesangial cells. These two cell types both express high l evels of cGK. KT5823 inhibited purified cGK, However, with both intact huma n platelets and rat mesangial cells, KT5823 failed to inhibit cGK-mediated serine 157 and serine 239 phosphorylation of VASP induced by nitric oxide, atrial natriuretic peptide, or the membrane-permeant cGMP analog 8-pCPT-cGM P, KT5823 enhanced 8-pCPT-cGMP-stimulated VASP phosphorylation in platelets and did not inhibit forskolin-stimulated VASP phosphorylation in either pl atelets or mesangial cells, In contrast H89, an inhibitor of both PKA. and cGK, clearly inhibited 8-pCPT-cGMP and forskolin-stimulated VASP phosphoryl ation in the two cell types. The data indicate that KT5823 inhibits purifie d cGK but does not affect a cGK-mediated response in the two different cell types expressing cGK I. These observations indicate that data that interpr et the effects of KT5823 in intact cells as the major or only criteria supp orting the involvement of cGK clearly need to be reconsidered.