Background

Soluble guanylyl cyclase (sGC) constitutes a family of enzymes that catalyses the cyclization of guanosine 5’-tri-phosphate (GTP) to guanosine 3’,5’-cyclic monophosphate (cGMP). The heterodimeric hemoprotein is activated by nitric oxide and mediates a wide range of physiological effects like regulation of blood pressure and neuronal cell development. sGC is an important target for the treatment of cardiovascular diseases. In addition to the biosynthesis of cGMP, we recently showed that invitro and in presence of Mn2+ ions sGC also generates the cyclic purine nucleotides adenosine 3’,5’-cyclic monophosphate (cAMP), inosine 3’,5’-cyclic monophosphate (cIMP), and xanthosine 3’,5’-cyclic monophosphate (cXMP)[1]. For all purine nucleotides, a second low-affinity site was described[1]. Moreover, sGC shows a pyrimidinylyl cyclase activity for uridine 3’,5’-cyclic monophosphate (cUMP) and cytosine 3’,5’-cyclic monophosphate (cCMP). In this case, no second binding site could be identified[1]. Lastly, no formation of the cyclic desoxyribonucleotide thymidine 3’,5’-cyclic monophosphate (cTMP) was detected[1]. Materials and methods For in vitro assays and determination of Michaelis-Men-ten kinetic parameters highly purified recombinant sGC from rat (a1b1) was activated by sodium nitroprusside (100 µM). sGC (0.1-20 ng/tube) was incubated at 37°C with 3 mM MnCl2 or MgCl2 and various concentrations of nucleoside 5’-triphosphates (NTPs). Samples were stopped by heating at 95°C. Concentrations of cyclic nucleotides were determined by HPLC-MS/MS. The in vivo assays were performed using transiently a1b1-transfected HEK 293 and endogenously sGC-expressing rat fetal lung fibroblast RFL-6 cells[2]. Cells were stimulated with SNP (100 µM) with or without phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 µM). After defined times medium was removed and cell metabolism was stopped by addition of organic solvent and by heating at 98°C. After centri-fugation clear supernatant was taken for quantitation of cyclic nucleotides by HPLC-MS/MS