The Protein Kinase C Inhibitor Gö6976 Blocks C-Type Natriuretic Peptide Activation of Guanylyl Cyclase B

Additional contributors: Jerid W. Robinson; Lincoln R. Potter (faculty mentor).C-type natriuretic peptide (CNP) activates the transmembrane guanylyl cyclase natriuretic peptide receptor-B (NPR-B/GC-B), which stimulates cGMP synthesis and mediates long bone growth, vasorelaxation, and axonal guidance. This study characterizes the effects of the widely used protein kinase C inhibitor, Gö6976, on NPR-B guanylyl cyclase activity. In membrane guanylyl cyclase assays, Gö6976 inhibited CNP-dependent guanylyl cyclase activity, but had no effect on detergent dependent guanylyl cyclase activities, suggesting that the effect of Gö6976 is on the CNP-dependent activation process, not on the formation of the catalytic site. Gö6976 inhibited mutant forms of NPR-B lacking known phosphorylation sites in a manner similar to in the wild type receptor, consistent with a process that does not require changes in receptor phosphorylation status. Nanomolar concentrations of Gö6976 inhibited NPR-B activity in 293 cells sensitive to phorbol ester-dependent inhibition of NPR-B (293PMA), whereas micromolar amounts of Gö6976 were required to produce a less marked effect on NPR-B activity in 293 cells that are moderately sensitive to PMA inhibition (293T), and no significant inhibition by Gö6976 was observed in a line of 293 cells that are not sensitive to phorbol ester-dependent inhibition (293neo). The differential inhibition of NPR-B activity in these cell lines suggests that cellular environment plays a critical role in the mechanism of Gö6976-dependent inhibition of NPR-B. Finally, direct addition of Gö6976 to crude 293T cell membrane also reduced CNP-dependent guanylyl cyclase activity, indicating that the preservation of cellular machinery is not required for the inhibitory process