Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells

doi:10.1016/j.bbrc.2013.12.108

Biochemical and Biophysical Research Communications

Volume 443, Issue 4, 24 January 2014, Pages 1195-1199

https://doi.org/10.1016/j.bbrc.2013.12.108 Get rights and content

Highlights

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The presence of cCMP and cUMP in intact cells is unambiguously documented.
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Soluble guanlyl cyclase generates cCMP and cUMP in intact cells.
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cGMP, cCMP and cUMP show different regulatory patterns.
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Mn²⁺ is a physiological cofactor for soluble guanylyl cyclase.

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 Mn²⁺ but not Mg²⁺, 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 Mn²⁺ is a physiological sGC cofactor.

Graphical abstract

Introduction

The cyclic purine nucleotides cAMP and cGMP are established second messengers regulating numerous physiological processes, such as relaxation of smooth muscle cells, differentiation and neurotransmission [1], [2]. The existence of the cyclic pyrimidine nucleotides cCMP and cUMP in tissues had been postulated [3], [4]. Moreover, a specific cytidylyl cyclase and a cCMP-degrading PDE was claimed [5], [6]. However, previous methods used to demonstrate the occurrence and generation of cCMP lacked selectivity and sensitivity, resulting in controversial discussion [7]. As a result, very little research has been conducted in the cCMP and cUMP field over the past three decades.

Recently, cCMP and cUMP have been shown to activate PKA with lower potency than, but similar efficacy as, cAMP [8]. cCMP and cUMP are less potent and effective activators of PKG than cGMP [8]. In contrast to cAMP, cGMP and cUMP, cCMP is not cleaved by several human recombinant PDEs [9], pointing to different roles of the various cNMPs in signal transduction. The membrane-permeable cCMP analog dibutyryl-cCMP induces vascular smooth muscle relaxation via PKG [10]. Moreover, cCMP and cUMP partially activate the ion channels HCN2 and 4 in recombinant cells and native cardiomyocytes [11]. Furthermore, by using radiometric-, HPLC- and MS approaches, purified bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis were shown to produce cCMP and cUMP [12]. Lastly, using a highly sensitive and specific HPLC–MS/MS method, purified sGC α₁β₁ has been shown to produce cCMP and cUMP NO-dependently [13]. Based on these data we developed the hypothesis that cCMP and cUMP play distinct roles as second messenger [14]. However, since cCMP- and cUMP formation by purified sGC occurred only in the presence of Mn²⁺, the physiological relevance of cyclic pyrimidine nucleotide formation by sGC remained unclear. Here, we show that sGC catalyzes cCMP- and cUMP formation also in intact cells.

Section snippets

Materials

SNP, IBMX, DEA/NO, ODQ and M7403 medium were purchased from Sigma–Aldrich (Seelze, Germany). Ham’s F12, DMEM high glucose 4.5 g/L, penicillin, streptomycin, l-glutamine, and Dulbecco’s PBS was purchased from PAA (Pasching, Austria). Fetal bovine serum was obtained from Lonza (Verviers, Belgium). FuGene was from Roche (Mannheim, Germany) and Zeocin was from Invitrogen (Darmstadt, Germany). HPLC-grade acetonitrile, methanol, and water were supplied by Baker (Deventer, The Netherlands). Tenofovir

Identification of cCMP and cUMP in intact cells

In addition to the well-known second messengers cAMP and cGMP we detected the cyclic pyrimidine nucleotides cCMP and cUMP at substantial concentrations in untransfected HEK293 cells and RFL-6 cells using HPLC–MS/MS technology (Table S2). The cNMP concentrations and the cNMP ratios in the two cell types were different, pointing to specific functions of each cNMP (Tables S2 and S3). In HEK293 cells cultured for prolonged time periods (48 h), basal cNMP concentrations decreased, with cCMP and cUMP

Discussion

Early studies in the mid 1980s tentatively identified cCMP in tissues using fast atom bombardment MS [3], [4], but specificity and sensitivity of the method are insufficient for unequivocal identification as compared to modern MS analytical methods. Specificity of cCMP antibodies is also problematic [19]. In the present study, we took advantage of the most advanced HPLC–MS/MS and HPLC–MS/TOF methods currently available to detect cNMPs. In this study, the cyclic pyrimidine nucleotides cCMP and

Acknowledgments

We thank Mrs. Annette Garbe for expert technical assistance and Dr. Ulrich Förstermann (University of Mainz, Germany) for providing RFL-6 cells. This work was supported by grants of the Deutsche Forschungsgemeinschaft to R.S.

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¹: These authors contributed equally to this work.

²: Present address: Evotec AG, Essener Bogen 7, D-22419 Hamburg, Germany.

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Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Identification of cCMP and cUMP in intact cells

Discussion

Acknowledgments

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

FEBS Lett.

FEBS Lett.

J. Biol. Chem.

Nitric Oxide

J. Proteomics

Pharmacol. Ther.

Int. J. Biochem. Cell Biol.

Capturing cyclic nucleotides in action: snapshots from crystallographic studies

Nat. Rev. Mol. Cell. Biol.

CGMP regulated protein kinases (cGK)

Handb. Exp. Pharmacol.

Extraction, purification and identification of cytidine 3′,5′-cyclic monophosphate from rat tissues

Biochem. J.

Extraction, purification, identification and metabolism of 3′,5-cyclic UMP, 3′,5′-cyclic IMP and 3′,5′-cyclic dTMP from rat tissues

Biochem. J.

Cytidine 3′,5′-monophosphate (cyclic CMP) formation in mammalian tissues

Science

Cytidylyl and uridylyl cyclase activity of Bacillus anthracis edema factor and Bordetella pertussis CyaA

Biochemistry