title: Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells.
abstract: Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α1β1 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 α1β1 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.
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Electronic address: baehre.heike@mh-hannover.de.
authors.forename: Heike
authors.initials: H
authors.lastname: Bähre
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Electronic address: kerstin.danker@evotec.com.
authors.forename: Kerstin Y
authors.initials: KY
authors.lastname: Danker
authors.affiliation: Institute of Cardiovascular Research, Bayer Healthcare, D-42096 Wuppertal, Germany. Electronic address: johannes-peter.stasch@bayer.com.
authors.forename: Johannes-Peter
authors.initials: JP
authors.lastname: Stasch
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany; Core Unit Metabolomics, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Electronic address: kaever.volkhard@mh-hannover.de.
authors.forename: Volkhard
authors.initials: V
authors.lastname: Kaever
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. Electronic address: seifert.roland@mh-hannover.de.
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D003563:Cyclic CMP / Q000096:biosynthesis; D006152:Cyclic GMP / Q000096:biosynthesis; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008345:Manganese / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D009712:Nucleotides, Cyclic / Q000096:biosynthesis; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems; D000071756:Soluble Guanylyl Cyclase; D014162:Transfection; D014542:Uridine Monophosphate / Q000096:biosynthesis
chemical_list: D020030:Nitric Oxide Donors; D009712:Nucleotides, Cyclic; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D009599:Nitroprusside; D009569:Nitric Oxide; D003563:Cyclic CMP; C045338:cyclic 3',5'-uridine monophosphate; D008345:Manganese; D000242:Cyclic AMP; D014542:Uridine Monophosphate; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP
keywords: Cyclic CMP; Cyclic GMP; Cyclic UMP; Manganese; Nitric oxide; Soluble guanylyl cyclase