Soluble adenylyl cyclase accounts for high basal cCMP and cUMP concentrations in HEK293 and B103 cells.

Intact HEK293 cells and B103 neuroblastoma cells possess high basal concentrations of the established second messengers cAMP and cGMP and of the emerging second messengers cCMP and cUMP. We asked the question which nucleotidyl cyclase accounts for the high basal cNMP concentrations. Activators and inhibitors of soluble guanylyl cyclase had no major effects on cNMPs, and the activator of membranous adenylyl cyclase forskolin increased only cAMP. Addition of bicarbonate to medium increased, whereas removal of bicarbonate decreased levels of all four cNMPs. The inhibitor of soluble adenylyl cyclase, 2-(1H-benzo[d]imidazol-2-ylthio)-N'-(5-bromo-2-hydroxybenzylidene) propanehydrazide (KH7), reduced bicarbonate-stimulated cNMPs. In conclusion, bicarbonate-stimulated soluble adenylyl cyclase plays an important role in the regulation of basal cellular cNMP levels, most notably cCMP and cUMP.

Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells.

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α1ß1 has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn(2+) but not Mg(2+), the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α1ß1 and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC-tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC-time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn(2+) is a physiological sGC cofactor.