Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

Background: Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one alpha and one beta subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results: Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and beta(1) subunit protein levels. Both sGC activity and beta(1) subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases beta(1) subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in beta(1) subunits. Conclusions: These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing beta(1) subunit stability.