Erich H. Schneider

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.

Analysis of Histamine Receptor Knockout Mice in Models of Inflammation

The diverse functions of histamine are mediated by four specific histamine receptor subtypes, which belong to the family of G-protein-coupled receptors. Here, we summarize data obtained with histamine-deficient l-histidine decarboxylase knockout and histamine receptor subtype knockout mice in inflammation models. Advantages and disadvantages of the knockout approaches compared with pharmacologic approaches are discussed critically. Due to many controversial data it is very difficult to draw clear-cut conclusions from the data provided in the literature. Thus, the published studies highlight the complexity of histamine function in inflammation and the need for much more systematic experimental work.

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.

Report on the Third Symposium “cCMP and cUMP as New Second Messengers”

The cyclic pyrimidine nucleotides cytidine 3′,5′-cyclic monophosphate (cCMP) and uridine 3′,5′-cyclic monophosphate (cUMP) have been unequivocally identified in mammalian cells using the most advanced mass spectrometry methods. On October 10, 2014, leading experts in the field met at the Hannover Medical School, Hannover, Germany, to discuss the latest findings in this emerging field of research. Generators, effectors, biological functions, inactivation mechanisms, and model systems for cCMP and cUMP were discussed. Pseudomonas aeruginosa nucleotidyl cyclase toxin ExoY, effectively producing cUMP, was a central topic of the meeting. cCMP and cUMP fulfill the criteria for second messengers. Future research directions in the field will include the identification of specific effector proteins of cCMP and cUMP, new cCMP- and cUMP-generating bacterial toxins, the analysis of new model organisms such as the zebra fish, and elucidation of the function of other noncanonical cyclic nucleotides such as inosine 3′,5′-cyclic monophosphate (cIMP).

Altered histamine neurotransmission in HPRT-deficient mice

Lesch-Nyhan syndrome (LNS) is an X-chromosomal disorder with congenital deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) as underlying defect. We determined the concentrations of dopamine, histamine and their metabolites in brains of HPRT knockout mice, which serve as an animal model for LNS, and compared the results to those obtained from wild-type controls. Analyses were performed by high performance liquid chromatography (HPLC)-coupled tandem mass spectrometry (MS/MS). Besides a decrease of dopamine and 3-methoxytyramine (3-MT) concentrations in the cerebral hemisphere, HPRT-deficient mice also exhibited significantly reduced 1-methylhistamine (1-MH) and 1-methylimidazole-4-acetic acid (1-MI4AA) concentrations in the brain hemisphere and medulla. Moreover, the amount of 1-MI4AA was significantly decreased in the cerebellum. Our findings show that neuronal perturbations caused by HPRT deficiency are not restricted to the dopamine system but also affect histaminergic neurotransmission. These new insights into the brain metabolism of an LNS mouse model may help to find new therapeutic strategies to improve the quality of life of LNS patients.

Flow cytometric analysis with a fluorescently labeled formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2 receptor

The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3′,5′-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca2+]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca2+]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.

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.

Neurotransmitter and their metabolite concentrations in different areas of the HPRT knockout mouse brain

Lesch-Nyhan syndrome (LNS) is characterized by uric acid overproduction and severe neurobehavioral symptoms, such as recurrent self-mutilative behavior. To learn more about the pathophysiology of the disease, we quantified neurotransmitters and their metabolites in the cerebral hemisphere, cerebellum and the medulla oblongata of HPRT knockout mice, an animal model for LNS, in comparison to the corresponding wild-type. Our analyses included l-glutamate, 4-aminobutanoic acid (GABA), acetylcholine, serotonin, 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine, l-normetanephrine, epinephrine and l-metanephrine and were conducted via high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS). Among these neurotransmitter systems, we did not find any abnormalities in the HPRT knockout mouse brains. On one side, this might indicate that HPRT deficiency most severely affects dopamine signaling, while brain functioning based on other neurotransmitters is more or less spared. On the other hand, our findings may reflect a compensating mechanism for impaired purine salvage that protects the brain in HPRT-deficient mice but not in LNS patients.

Fishing for elusive cCMP-degrading phosphodiesterases

Background The literature of the past four decades contains several scattered reports that demonstrate the existence of two different 3’,5’-cCMP degrading enzymes with distinct substrate affinity and specificity. The so-called “cCMPspecific” PDE was isolated from disrupted L-1210 leukemia cells [1] and from rat liver [2,3], shows an extremely high 3’,5’-cCMP KM value of up to 9 mM and seems to be activated by iron [1]. This enzyme prefers 3’,5’-cCMP over other cyclic nucleotides. The other enzyme is called “multifunctional cCMP PDE” and was isolated from pig liver [3-5], rat liver [6,7] and human liver [8]. This enzyme shows a 3’,5’-cCMP KM value in the range of 100-300 μM and is additionally degrading 2’,3’-cCMP as well as the 2’,3’and 3’,5’-isomers of cUMP, cAMP, cGMP and cIMP. The multifunctional cCMP PDE is inhibited by inorganic phosphate and AMP. Both enzymes are calmodulin-insensitive, IBMX-resistant and show a low molecular weight of <35 kDa, which is uncommon for the “classic” phosphodiesterases. However, the exact amino acid sequence and identity of these proteins remain elusive. Our project aims at a clear and unequivocal determination of amino acid sequence and identity of these elusive cCMP-degrading enzymes by analyzing and purifying the cCMP degrading activity of organ extracts and biological fluids (e.g. serum). Moreover, we re-investigate wellknown “classic” PDEs in enzymatic assays to identify a potential 3’,5’-cCMP degrading activity.

New perspectives for a well-known biogenic amine: mast cell-derived histamine as pathophysiological agent in vincristine-induced neuropathic pain

Neuropathic pain occurs in up to 10% of the general population (Binder and Baron 2016) and is treated by numerous medical drugs with very diverse mechanisms of action, e.g., anticonvulsants, tricyclic antidepressants, or opioids (Dosenovic et al. 2017). The effectiveness of such therapies, however, is limited, and a maximum pain reduction of only 30–50% is to be expected (Binder and Baron 2016). One wellknown cause of neuropathic pain is cancer chemotherapy with cytostatic drugs like vincristine, cisplatin, oxaliplatin, paclitaxel, or bortezomib (Carozzi et al. 2015). This side effect often limits clinical applicability of such life-saving medications. Mast cell-derived mediators including histamine seem to play an important role in the pathophysiology of various forms of neuropathic pain (Chatterjea and Martinov 2015). Nothing is known, however, about the involvement of mast cells in chemotherapy-induced neuropathy. This situation prompted Jaggi et al. (2017) to perform the highly interesting study published in this issue of Naunyn Schmiedeberg’s Archives of Pharmacology. The authors explored the applicability of mast cell stabilizers as well as histamine H1R and H2R antagonists for the treatment of vincristine-induced neuropathic pain in a carefully designed 28-day experiment using Wistar rats as model organisms. Neuropathic pain was induced by treating the animals with ten doses of vincristine (100 μg/kg) from day 0 to day 11 (two blocks of 5 days interrupted by a 2-day break). One group was treated with vincristine only (positive control). Six groups of animals received vincristine in combination with sodium cromoglycate (10 or 20 mg/kg), promethazine (50 or 100 μg/kg), and ranitidine (20 or 40 mg/kg). An untreated control group was run in parallel. Pain sensitivity was evaluated on day 0 (baseline), day 14, and day 28 by systematically assessing mechanical hyperalgesia (pin prick test) and hypersensitivity to cold (acetone drop test) and hot (hot plate test) thermal stimuli. All experimenters were blinded to the treatment groups. This guarantees objectivity of the study and compensates for sources of uncertainties like human observerbased evaluation of video recordings or the use of a noncalibrated stimulus in the pinprick test. The authors report a significant reduction of vincristineinduced neuropathic pain by all three therapeutic agents in all three experimental setups for pain assessment. It may, however, be argued that the effect of promethazine is partially due to sedative effects. Moreover, promethazine is not selective for the histamine H1R but also antagonizes human and guinea pig histamineH2 receptorswith aKB value in the submicromolar range (Appl et al. 2012) and shows prominent anticholinergic properties. Furthermore, promethazine binds to other biogenic amine receptors, e.g., to the dopamine D2 receptor (Seeman et al. 1985) and to serotonin 5-HT2 receptor subtypes (Fiorella et al. 1995). Thus, the observed action of promethazine on neuropathic pain may be partially due to off-target effects. The authors therefore recommend the use of H1R antagonists with higher receptor selectivity in future studies. By contrast, sedation is not known to be a common side effect of sodium cromoglycate and ranitidine, and both compounds do not show as many off-target effects as promethazine. Thus, the amelioration of neuropathic pain by sodium cromoglycate or ranitidine is very likely due to mast cell stabilization or H2R antagonism, respectively. Nevertheless, the authors recommend that future studies should confirm their results * Erich H. Schneider schneider.erich@mh-hannover.de