Heike Bähre

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 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.

ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation

In addition to the well known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. Soluble guanylyl cyclase and soluble adenylyl cyclase produce all four cNMPs. Several bacterial toxins exploit mammalian cyclic nucleotide signaling. The type III secretion protein ExoY from Pseudomonas aeruginosa induces severe lung damage and effectively produces cGMP. Here, we show that transfection of mammalian cells with ExoY or infection with ExoY-expressing P. aeruginosa not only massively increases cGMP but also cUMP levels. In contrast, the structurally related CyaA from Bordetella pertussis and edema factor from Bacillus anthracis exhibit a striking preference for cAMP increases. Thus, ExoY is a nucleotidyl cyclase with preference for cGMP and cUMP production. The differential effects of bacterial toxins on cNMP levels suggest that cUMP plays a distinct second messenger role.

From canonical to non-canonical cyclic nucleotides as second messengers: pharmacological implications.

This review summarizes our knowledge on the non-canonical cyclic nucleotides cCMP, cUMP, cIMP, cXMP and cTMP. We place the field into a historic context and discuss unresolved questions and future directions of research. We discuss the implications of non-canonical cyclic nucleotides for experimental and clinical pharmacology, focusing on bacterial infections, cardiovascular and neuropsychiatric disorders and reproduction medicine. The canonical cyclic purine nucleotides cAMP and cGMP fulfill the criteria of second messengers. (i) cAMP and cGMP are synthesized by specific generators, i.e. adenylyl and guanylyl cyclases, respectively. (ii) cAMP and cGMP activate specific effector proteins, e.g. protein kinases. (iii) cAMP and cGMP exert specific biological effects. (iv) The biological effects of cAMP and cGMP are terminated by phosphodiesterases and export. The effects of cAMP and cGMP are mimicked by (v) membrane-permeable cyclic nucleotide analogs and (vi) bacterial toxins. For decades, the existence and relevance of cCMP and cUMP have been controversial. Modern mass-spectrometric methods have unequivocally demonstrated the existence of cCMP and cUMP in mammalian cells. For both, cCMP and cUMP, the criteria for second messenger molecules are now fulfilled as well. There are specific patterns by which nucleotidyl cyclases generate cNMPs and how they are degraded and exported, resulting in unique cNMP signatures in biological systems. cNMP signaling systems, specifically at the level of soluble guanylyl cyclase, soluble adenylyl cyclase and ExoY from Pseudomonas aeruginosa are more promiscuous than previously appreciated. cUMP and cCMP are evolutionary new molecules, probably reflecting an adaption to signaling requirements in higher organisms.

Delineating the Role of Histamine-1- and -4-Receptors in a Mouse Model of Th2-Dependent Antigen-Specific Skin Inflammation

Background Histamine drives pruritus in allergic skin diseases which clinically constitutes a most disruptive symptom. Skin pathology in allergic skin diseases is crucially influenced by different T-helper subsets. However, the contribution of different histamine-receptors to T-helper cell dependent skin pathology has not been definitively answered. Models which can specifically address the functional role of T-helper subsets and the mediators involved are therefore valuable to gain further insights into molecular pathways which contribute to allergic skin disease. They might also be helpful to probe amendable therapeutic interventions such as histamine-receptor antagonism. Objective Establishing an adoptive transfer model for antigen-specific Th cells, we aimed to delineate the role of histamine H1- and H4-receptors in Th2-dependent skin inflammation. Methods In-vitro differentiated and OVA primed Th2 cells were adoptively transferred into congenic recipient mice. In vivo treatment with specific histamine H1- and H4-receptor antagonists was performed to analyze the contribution of these histamine-receptors to Th2-dependent skin pathology in our model. Analysis four days after epicutaneous challenge comprised skin histology, flow cytometric detection of transferred T-helper cells and analysis of antigen-cytokine profiles in skin-draining lymph nodes. Results Use of specific H1- and H4-receptor antagonists revealed a crucial role for H1- and H4-receptors for Th2 migration and cytokine secretion in a Th2-driven model of skin inflammation. While H1- and H4-receptor antagonists both reduced Th2 recruitment to the site of challenge, local cytokine responses in skin-draining lymph nodes were only reduced by the combined application of H1- and H4-receptor antagonists and mast cell counts remained altogether unchanged by either H1R-, H4R- or combined antagonism. Conclusion Our model demonstrates a role for H1- and H4-receptors in Th2 cell infiltration and cytokine secretion in allergic skin diseases and suggests further studies to evaluate these findings for therapeutic approaches.

PDE7A1 hydrolyzes cCMP

The degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3′,5′‐cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly‐sensitive HPLC–MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST‐tagged truncated PDE7A1 revealed a cCMP K M value of 135 ± 19 μM. The V max for cCMP hydrolysis reached 745 ± 27 nmol/(min mg), which is about 6‐fold higher than the corresponding velocity for adenosine 3′,5′‐cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high‐speed and low‐affinity PDE for cCMP.

Non-targeted metabolomics by high resolution mass spectrometry in HPRT knockout mice.

AIMS Lesch-Nyhan disease (LND) is characterized by hyperuricemia as well as neurological and neuropsychiatric symptoms including repetitive self-injurious behavior. Symptoms are caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a result of a mutation on the X chromosome. To elucidate the pathophysiology of LND, we performed a metabolite screening for brain and serum extracts from HPRT knockout mice as an animal model for LND. MAIN METHODS Analyses were performed by high performance liquid chromatography (HPLC)-coupled quadrupole time-of-flight mass spectrometry (QTOF-MS). KEY FINDINGS In brain extracts, we found six metabolites with significantly different contents in wild-type and HPRT-deficient mice. Two compounds we could identify as 5-aminoimidazole-4-carboxamide ribotide (AICAR) and 1-methylimidazole-4-acetic acid (1-MI4AA). Whereas AICAR was accumulated in brains of HPRT knockout mice, 1-MI4AA was decreased in these mice. SIGNIFICANCE Both metabolites play a role in histidine metabolism and, as a consequence, histamine metabolism. AICAR, in addition, is part of the purine metabolism. Our findings may help to better understand the mechanisms leading to the behavioral phenotype of LND.

Analytical Methods for the Quantification of Histamine and Histamine Metabolites

The endogenous metabolite histamine (HA) is synthesized in various mammalian cells but can also be ingested from exogenous sources. It is involved in a plethora of physiological and pathophysiological processes. So far, four different HA receptors (H1R-H4R) have been described and numerous HAR antagonists have been developed. Contemporary investigations regarding the various roles of HA and its main metabolites have been hampered by the lack of highly specific and sensitive analytic methods for all of these analytes. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is the method of choice for identification and sensitive quantification of many low-molecular weight endogenous metabolites. In this chapter, different methodological aspects of HA quantification as well as recommendations for LC-MS/MS methods suitable for analysis of HA and its main metabolites are summarized.

cIMP: Synthesis, effector activation, inactivation and occurrence in biological systems

Background The soluble nitric oxide (NO)-stimulated guanylyl cyclase (sGC) uses GTP as a substrate to synthesize the secondary messenger cGMP [1]. However, sGC is not so stringent in terms of substrate-specificity and can also use ATP to produce yet another secondary messenger, cAMP [1]. Both cAMP and cGMP induce vasodilation. Recently, it has been proposed that under conditions of hypoxia, sGC predominantly produces cIMP to induce vasoconstriction [2]. Chemically, cGMP and cIMP are very closely related to each other, i.e. the only chemical difference between the two cyclic nucleotides is the missing amino group at the 2’-position of the purine ring (Fig. 1). How can such a small chemical difference between two molecules lead to opposite biological effects? To answer this question, we studied cIMP synthesis, effector activation, inactivation and biological occurrence.