Leukocyte chemotactic receptor Fpr1 protects against aging-related posterior subcapsular cataract formation.

Cataracts are a common consequence of aging; however, pathogenesis remains poorly understood. Here, we observed that after 3 months of age mice lacking the G protein-coupled leukocyte chemotactic receptor Fpr1 (N-formyl peptide receptor 1) began to develop bilateral posterior subcapsular cataracts that progressed to lens rupture and severe degeneration, without evidence of either systemic or local ocular infection or inflammation. Consistent with this, Fpr1 was detected in both mouse and human lens in primary lens epithelial cells (LECs), the only cell type present in the lens; however, expression was confined to subcapsular LECs located along the anterior hemispheric surface. To maximize translucency, LECs at the equator proliferate and migrate posteriorly, then differentiate into lens fiber cells by nonclassical apoptotic signaling, which results in loss of nuclei and other organelles, including mitochondria which are a rich source of endogenous N-formyl peptides. In this regard, denucleation and posterior migration of LECs were abnormal in lenses from Fpr1-/- mice, and direct stimulation of LECs with the prototypic N-formyl peptide agonist fMLF promoted apoptosis. Thus, Fpr1 is repurposed beyond its immunoregulatory role in leukocytes to protect against cataract formation and lens degeneration during aging.

Inactivation of Non-canonical Cyclic Nucleotides: Hydrolysis and Transport.

This chapter addresses cNMP hydrolysis by phosphodiesterases (PDEs) and export by multidrug resistance associated proteins (MRPs). Both mechanisms are well-established for the canonical cNMPs, cAMP, and cGMP. Increasing evidence shows that non-canonical cNMPs (specifically cCMP, cUMP) are also PDE and MRP substrates. Hydrolysis of cUMP is achieved by PDE 3A, 3B, and 9A, which possibly explains the cUMP-degrading activities previously reported for heart, adipose tissue, and brain. Regarding cCMP, the only known "conventional" (class I) PDE that hydrolyzes cCMP is PDE7A. Older reports describe cCMP-degrading PDE-like activities in mammalian tissues, bacteria, and plants, but the molecular identity of these enzymes is not clear. High K M and V max values, insensitivity to common inhibitors, and unusually broad substrate specificities indicate that these activities probably do not represent class I PDEs. Moreover, the older results have to be interpreted with caution, since the historical analytical methods were not as reliable as modern highly sensitive and specific techniques like HPLC-MS/MS. Besides PDEs, the transporters MRP4 and 5 are of major importance for cAMP and cGMP disposal. Additionally, both MRPs also export cUMP, while cCMP is only exported by MRP5. Much less data are available for the non-canonical cNMPs, cIMP, cXMP, and cTMP. None of these cNMPs has been examined as MRP substrate. It was shown, however, that they are hydrolyzed by several conventional class I PDEs. Finally, this chapter reveals that there are still large gaps in our knowledge about PDE and MRP activities for canonical and non-canonical cNMPs. Future research should perform a comprehensive characterization of the known PDEs and MRPs with the physiologically most important cNMP substrates.

Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutants in the Sf9 Cell Expression System.

A large problem of histamine receptor research is data heterogeneity. Various experimental approaches, the complex signaling pathways of mammalian cells, and the use of different species orthologues render it difficult to compare and interpret the published results. Thus, the four human histamine receptor subtypes were analyzed side-by-side in the Sf9 insect cell expression system, using radioligand binding assays as well as functional readouts proximal to the receptor activation event (steady-state GTPase assays and [35S]GTPγS assays). The human H1R was co-expressed with the regulators of G protein signaling RGS4 or GAIP, which unmasked a productive interaction between hH1R and insect cell Gαq. By contrast, functional expression of the hH2R required the generation of an hH2R-Gsα fusion protein to ensure close proximity of G protein and receptor. Fusion of hH2R to the long (GsαL) or short (GsαS) splice variant of Gαs resulted in comparable constitutive hH2R activity, although both G protein variants show different GDP affinities. Medicinal chemistry studies revealed profound species differences between hH1R/hH2R and their guinea pig orthologues gpH1R/gpH2R. The causes for these differences were analyzed by molecular modeling in combination with mutational studies. Co-expression of the hH3R with Gαi1, Gαi2, Gαi3, and Gαi/o in Sf9 cells revealed high constitutive activity and comparable interaction efficiency with all G protein isoforms. A comparison of various cations (Li+, Na+, K+) and anions (Cl-, Br-, I-) revealed that anions with large radii most efficiently stabilize the inactive hH3R state. Potential sodium binding sites in the hH3R protein were analyzed by expressing specific hH3R mutants in Sf9 cells. In contrast to the hH3R, the hH4R preferentially couples to co-expressed Gαi2 in Sf9 cells. Its high constitutive activity is resistant to NaCl or GTPγS. The hH4R shows structural instability and adopts a G protein-independent high-affinity state. A detailed characterization of affinity and activity of a series of hH4R antagonists/inverse agonists allowed first conclusions about structure/activity relationships for inverse agonists at hH4R. In summary, the Sf9 cell system permitted a successful side-by-side comparison of all four human histamine receptor subtypes. This chapter summarizes the results of pharmacological as well as medicinal chemistry/molecular modeling approaches and demonstrates that these data are not only important for a deeper understanding of HxR pharmacology, but also have significant implications for the molecular pharmacology of GPCRs in general.

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.

Regulation of motor function and behavior by atypical chemokine receptor 1.

Atypical Chemokine Receptor 1 (ACKR1), previously known as Duffy Antigen Receptor for Chemokines, stands out among chemokine receptors for high selective expression on cerebellar Purkinje neurons. Although ACKR1 ligands activate Purkinje cells in vitro, evidence for ACKR1 regulation of brain function in vivo is lacking. Here we demonstrate that Ackr1 (-/-) mice have markedly impaired balance and ataxia on a rotating rod and increased tremor when injected with harmaline, which induces whole-body tremor by activating Purkinje cells. Ackr1 (-/-) mice also exhibited impaired exploratory behavior, increased anxiety-like behavior and frequent episodes of marked hypoactivity under low-stress conditions. Surprisingly, Ackr1 (+/-) had similar behavioral abnormalities, indicating pronounced haploinsufficiency. The behavioral phenotype of Ackr1 (-/-) mice was the opposite of mouse models of cerebellar degeneration, and the defects persisted when Ackr1 was deficient only on non-hematopoietic cells. Together, the results suggest that normal motor function and behavior may partly depend on negative regulation of Purkinje cell activity by Ackr1.

The leukocyte chemotactic receptor FPR1 is functionally expressed on human lens epithelial cells.

BACKGROUND: Lens degeneration in Fpr1(-/-) mice prompted us to search for functional FPR1 expression directly on lens epithelial cells. RESULTS: FPR1 is functionally expressed on human lens epithelial cells but has atypical properties compared with hematopoietic cell FPR1. CONCLUSION: Lens epithelial cell FPR1 may be involved in development and maintenance of the lens. SIGNIFICANCE: This is the first link between non-hematopoietic expression of FPR1 and an ophthalmologic phenotype. Formyl peptide receptor 1 (FPR1) is a G protein-coupled chemoattractant receptor expressed mainly on leukocytes. Surprisingly, aging Fpr1(-/-) mice develop spontaneous lens degeneration without inflammation or infection (J.-L. Gao et al., manuscript in preparation). Therefore, we hypothesized that FPR1 is functionally expressed directly on lens epithelial cells, the only cell type in the lens. Consistent with this, the human fetal lens epithelial cell line FHL 124 expressed FPR1 mRNA and was strongly FPR1 protein-positive by Western blot and FACS. Competition binding using FPR1 ligands N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (Nle = Norleucine), formylmethionylleucylphenylalanine, and peptide W revealed the same profile for FHL 124 cells, neutrophils, and FPR1-transfected HEK 293 cells. Saturation binding with fluorescein-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys revealed ~2500 specific binding sites on FHL-124 cells (K(D) ~ 0.5 nm) versus ~40,000 sites on neutrophils (K(D) = 3.2 nm). Moreover, formylmethionylleucylphenylalanine induced pertussis toxin-sensitive Ca(2+) flux in FHL 124 cells, consistent with classic G(i)-mediated FPR1 signaling. FHL 124 cell FPR1 was atypical in that it resisted agonist-induced internalization. Expression of FPR1 was additionally supported by detection of the intact full-length open reading frame in sequenced cDNA from FHL 124 cells. Thus, FHL-124 cells express functional FPR1, which is consistent with a direct functional role for FPR1 in the lens, as suggested by the phenotype of Fpr1 knock-out mice.

Paradoxical stimulatory effects of the "standard" histamine H4-receptor antagonist JNJ7777120: the H4 receptor joins the club of 7 transmembrane domain receptors exhibiting functional selectivity.

The histamine H(4) receptor (H(4)R) is expressed in several cell types of the immune system and is assumed to play an important pro-inflammatory role in various diseases, including bronchial asthma, atopic dermatitis, and pruritus. Accordingly, H(4)R antagonists have been suggested to provide valuable drugs for the treatment of these diseases. Over the past decade, the indole derivative 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) has become the "standard" H(4)R antagonist and has been extensively used to assess the pathophysiological role of the H(4)R. However, the situation has now become more complicated by recent data (p. 749 and Naunyn Schmiedebergs Arch Pharmacol doi: 10.1007/s00210-011-0612-3) showing that JNJ7777120 can also activate ß-arrestin in a supposedly G(i)-protein-independent (pertussis toxin-insensitive) manner and that at certain H(4)R species orthologs, JNJ7777120 exhibits partial agonist efficacy with respect to G(i)-protein activation (steady-state high-affinity GTPase activity). These novel findings can be explained within the concept of functional selectivity or biased signaling, assuming unique ligand-specific receptor conformations with distinct signal transduction capabilities. Thus, great caution must be exerted when interpreting in vivo effects of JNJ7777120 as H(4)R antagonism. We discuss future directions to get out of the current dilemma in which there is no "standard" H(4)R antagonist available to the scientific community.

Reduced fear memory and anxiety-like behavior in mice lacking formylpeptide receptor 1.

N-formylpeptide receptor 1 (FPR1) is a G protein-coupled receptor that mediates pro-inflammatory chemotactic responses by phagocytic leukocytes to N-formylpeptides produced by bacteria or mitochondria. Mice lacking Fpr1 (Fpr1 (-/-) mice) have increased susceptibility to challenge with certain bacteria. FPR1 is also a receptor for annexin-1, which mediates the anti-inflammatory effects of glucocorticoids as well as negative feedback by glucocorticoids of the hypothalamic-pituitary-adrenocortical axis. However, homeostatic functions of FPR1 in the neuroendocrine system have not previously been defined. Here we show that in systematic behavioral testing Fpr1 (-/-) mice exhibited increased exploratory activity, reduced anxiety-like behavior, and impaired fear memory, but normal spatial memory and learning capacity. Consistent with this, the homeostatic serum level of corticosterone in Fpr1 (-/-) mice was significantly lower compared with wild-type mice. The data implicate Fpr1 in modulation of anxiety-like behavior and fear memory by regulating glucocorticoid production.

Structural requirements for inverse agonism and neutral antagonism of indole-, benzimidazole-, and thienopyrrole-derived histamine H4 receptor ligands.

The human histamine H(4) receptor (hH(4)R), coexpressed with Galpha(i2) and Gbeta(1)gamma(2) in Sf9 insect cells, is highly constitutively active, and thioperamide [THIO; N-cyclohexyl-4-(imidazol-4-yl)-1-piperidinecarbothioamide] is one of the most efficacious hH(4)R inverse agonists. High constitutive hH(4)R activity may have pathophysiological implications in which case inverse agonists may behave differently than neutral antagonists. To learn more about the structural requirements for hH(4)R inverse agonism, we investigated 25 compounds (indole, benzimidazole, and thienopyrrole derivatives) structurally related to the standard antagonist JNJ-7777120 [1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine]. We characterized the compounds in radioligand binding assays by using [(3)H]histamine ([(3)H]HA) and in steady-state GTPase assays in the presence (antagonist mode) and absence (inverse agonist mode) of the agonist HA, yielding the following results: 1) Twenty-two compounds were inverse agonists (efficacy: 15-62% of the THIO effect), and only three compounds (12%) showed neutral antagonism. Thus, inverse agonism is far more common than neutral antagonism. 2) The inverse agonistic efficacy of the R5-monosubstituted indole-derived compounds increased with the volume of R5. R5 may interact with Trp(6.48) of the rotamer toggle switch and stabilize the inactive receptor conformation. 3) A subset of compounds showed large differences between the K(i) value from [(3)H]HA competition binding and the EC(50) value from steady-state GTPase assays, whereas the K(b) values were closer to the K(i) values. Thus, the two-state model should be extended to a model comprising a constitutively active hH(4)R state, which can be discriminated by inverse agonists from a structurally distinct HA-stabilized active state.

Impact of the DRY motif and the missing "ionic lock" on constitutive activity and G-protein coupling of the human histamine H4 receptor.

It is assumed that many G protein-coupled receptors (GPCRs) are restrained in an inactive state by the "ionic lock," an interaction between an arginine in transmembrane domain (TM) 3 (R3.50) and a negatively charged residue in TM6 (D/E6.30). In the human histamine H4 receptor (hH4R), alanine is present in position 6.30. To elucidate whether this mutation causes the high constitutive activity of hH4R, we aimed to reconstitute the ionic lock by constructing the A6.30E mutant. The role of R3.50 was investigated by generating hH4R-R3.50A. Both mutants were expressed alone or together with Galpha(i2) and Gbeta1gamma2 in Sf9 cells and characterized in GTPase, 35S-labeled guanosine 5'-[gamma-thio]triphosphate binding, and high-affinity agonist binding assays. Unexpectedly, compared with hH4R, hH4R-A6.30E showed only nonsignificant reduction of constitutive activity and G protein-coupling efficiency. The KD of [3H]histamine was unaltered. By contrast, hH4R-R3.50A did not stimulate G proteins. Thioperamide affinity at hH(4)R-R3.50A was increased by 300 to 400%, whereas histamine affinity was reduced by approximately 50%. A model of the active hH4R state in complex with the Galpha(i2) C terminus was compared with the crystal structures of turkey beta1 and human beta2 adrenoceptors. We conclude that 1) constitutive activity of hH4R is facilitated by the salt bridge D5.69-R6.31 rather than by the missing ionic lock, 2) Y3.60 may form alternative locks in active and inactive GPCR states, 3) R3.50 is crucial for hH4R-G protein coupling, and 4) hH4R-R3.50A represents an inactive state with increased inverse agonist and reduced agonist affinity. Thus, the ionic lock, although stabilizing the inactive rhodopsin state, is not generally important for all class A GPCRs.

Apoptotic and anti-proliferative effect of guanosine and guanosine derivatives in HuT-78 T lymphoma cells.

The effects of 100 µM of 3',5'-cGMP, cAMP, cCMP, and cUMP as well as of the corresponding membrane-permeant acetoxymethyl esters on anti-CD3-antibody (OKT3)-induced IL-2 production of HuT-78 cutaneous T cell lymphoma (Sézary lymphoma) cells were analyzed. Only 3',5'-cGMP significantly reduced IL-2 production. Flow cytometric analysis of apoptotic (propidium iodide/annexin V staining) and anti-proliferative (CFSE staining) effects revealed that 3',5'-cGMP concentrations > 50 µM strongly inhibited proliferation and promoted apoptosis of HuT-78 cells (cultured in the presence of αCD3 antibody). Similar effects were observed for the positional isomer 2',3'-cGMP and for 2',-GMP, 3'-GMP, 5'-GMP, and guanosine. By contrast, guanosine and guanosine-derived nucleotides had no cytotoxic effect on peripheral blood mononuclear cells (PBMCs) or acute lymphocytic leukemia (ALL) xenograft cells. The anti-proliferative and apoptotic effects of guanosine and guanosine-derived compounds on HuT-78 cells were completely eliminated by the nucleoside transport inhibitor NBMPR (S-(4-Nitrobenzyl)-6-thioinosine). By contrast, the ecto-phosphodiesterase inhibitor DPSPX (1,3-dipropyl-8-sulfophenylxanthine) and the CD73 ecto-5'-nucleotidase inhibitor AMP-CP (adenosine 5'-(α,ß-methylene)diphosphate) were not protective. We hypothesize that HuT-78 cells metabolize guanosine-derived nucleotides to guanosine by yet unknown mechanisms. Guanosine then enters the cells by an NBMPR-sensitive nucleoside transporter and exerts cytotoxic effects. This transporter may be ENT1 because NBMPR counteracted guanosine cytotoxicity in HuT-78 cells with nanomolar efficacy (IC50 of 25-30 nM). Future studies should further clarify the mechanism of the observed effects and address the question, whether guanosine or guanosine-derived nucleotides may serve as adjuvants in the therapy of cancers that express appropriate nucleoside transporters and are sensitive to established nucleoside-derived cytostatic drugs.

High constitutive activity and a G-protein-independent high-affinity state of the human histamine H(4)-receptor.

The human histamine H(4)-receptor (hH(4)R) is expressed in mast cells and eosinophils and mediates histamine (HA)-induced chemotaxis via G(i)-proteins. For a detailed investigation of hH(4)R/G(i)-protein interaction, we coexpressed the hH(4)R with Galpha(i2) and Gbeta(1)gamma(2) as well as an hH(4)R-Galpha(i2) fusion protein with Gbeta(1)gamma(2) in Sf9 insect cells. The agonist radioligand [(3)H]HA showed a K(D) value of approximately 10 nM at hH(4)R and hH(4)R-Galpha(i2). The high-affinity states of hH(4)R and hH(4)R-Galpha(i2) were insensitive to guanosine 5'-[gamma-thio]triphosphate (GTPgammaS). The affinity of [(3)H]HA for hH(4)R was retained in the absence of mammalian G(i)-proteins. In steady-state GTPase- and [(35)S]GTPgammaS-binding assays, hH(4)R exhibited high constitutive activity and uncommon insensitivity to Na(+). Thioperamide (THIO) was only a partial inverse agonist. Addition of HA or THIO to baculovirus-infected (hH(4)R + Galpha(i2) + Gbeta(1)gamma(2)) Sf9 cells increased the B(max) in [(3)H]HA binding, but not in immunoblots, suggesting conformational instability and ligand-induced stabilization of membrane-integrated hH(4)R. No effect was observed on hH(4)R-Galpha(i2) expression, neither in [(3)H]HA binding nor in immunoblot. However, the expression level of hH(4)R-Galpha(i2) was consistently higher compared to hH(4)R, suggesting chaperone-like or stabilizing effects of Galpha(i2) on hH(4)R. In 37 degrees C stability assays, HA stabilized hH(4)R, and THIO even restored misfolded [(3)H]HA binding sites. Inhibition of hH(4)R glycosylation by tunicamycin reduced the [(3)H]HA binding B(max) value. In conclusion, (i) hH(4)R shows high constitutive activity and structural instability; (ii) hH(4)R shows a G-protein-independent high-affinity state; (iii) hH(4)R conformation is stabilized by agonists, inverse agonists and G-proteins; (iv) hH(4)R glycosylation is essential for cell-surface expression of intact hH(4)R.

2,4-Diaminopyrimidines as histamine H4 receptor ligands--Scaffold optimization and pharmacological characterization.

The human histamine H(4) receptor (hH(4)R) is a promising new target in the therapy of inflammatory diseases and disorders of the immune system. For the development of new H(4)R antagonists a broad ligand-based virtual screening was performed resulting in two hits. The dissection of their common annelated aromatic core into its heteromonocyclic components showed that 2,4-diaminopyrimidine is a potent hH(4)R affinity scaffold, which was comprehensively investigated. Structure-activity relationship studies revealed that slight structural changes evoke extensive differences in functional activities and potencies: while o- and p-substituted benzyl amines mainly showed partial agonism, m-substituted and rigidified ones exhibited inverse agonist efficacy.

Hydrolysis of the non-canonical cyclic nucleotide cUMP by PDE9A: kinetics and binding mode.

The non-canonical cyclic nucleotide cUMP and the phosphodiesterase PDE9A both occur in neuronal cells. Using HPLC-coupled tandem mass spectrometry, we characterized the kinetics of PDE9A-mediated cUMP hydrolysis. PDE9A is a low-affinity and high-velocity enzyme for cUMP (Vmax = ~ 6 µmol/min/mg; Km = ~ 401 µM). The PDE9 inhibitor BAY 73-6691 inhibited PDE9A-catalyzed cUMP hydrolysis (Ki = 590 nM). Docking studies indicate two H-bonds between the cUMP uridine moiety and Gln453/Asn405 of PDE9A. By contrast, the guanosine moiety of cGMP forms three H-bonds with Gln453. cCMP is not hydrolyzed at a concentration of 3 µM, but inhibits the PDE9A-catalyzed cUMP hydrolysis at concentrations of 100 µM or more. The probable main reason is that the cytosine moiety cannot act as H-bond acceptor for Gln453. A comparison of PDE9A with PDE7A suggests that the preference of the former for cGMP and cUMP and of the latter for cAMP and cCMP is due to stabilized alternative conformations of the side chain amide of Gln453 and Gln413, respectively. This so-called glutamine switch is known to be involved in the regulation of cAMP/cGMP selectivity of some PDEs.

cUMP hydrolysis by PDE3B.

Previous results indicate that the phosphodiesterase PDE3B hydrolyzes cUMP. Also, almost 50 years ago, cUMP-hydrolytic activity was observed in rat adipose tissue. We intended to characterize the enzyme kinetics of PDE3B-mediated cUMP hydrolysis, to determine the PDE3B binding mode of cUMP, and to analyze cUMP hydrolysis in adipocyte preparations. Educts (cNMPs) and products (NMPs) of the PDE reactions as well as intracellular cNMPs were quantitated by HPLC-coupled tandem mass spectrometry. PDE3B expression was determined by qPCR and Western blot. Docking studies were performed with the PDE3B crystal structure PDB ID 1SO2 (complex with a dihydropyridazine inhibitor). PDE3B hydrolyzed cUMP (Km ~ 550 µM, Vmax ~ 76 µmol/min/mg) and cAMP (Km ~ 0.7 µM, Vmax ~ 4.3 µmol/min/mg) in a milrinone (PDE3-selective inhibitor)-sensitive manner (Ki for inhibition of cUMP hydrolysis: 205 nM). cUMP forms one hydrogen bond with PDE3B (uracil 3-NH with side chain oxygen of Q988). Two hydrogen bonds stabilize cAMP binding. cCMP does not interact with PDE3B. Possibly, the cytosine base cannot form hydrogen bonds with PDE3B, and the 4-NH2 group clashes with L987 of the enzyme. Adipocyte differentiation of 3T3-L1 MBX cells increased mRNA of PDE3B, but not of PDE3A. Significant amounts of cUMP were detected in differentiated and undifferentiated 3T3-L1 MBX cells. 3T3-L1 MBX adipocyte lysates and rat epididymal adipose tissue membranes contained milrinone-sensitive cUMP-hydrolytic activity. PDE3B is a low-affinity and high-velocity phosphodiesterase for cUMP. The cUMP-hydrolyzing activity described almost 50 years ago for rat adipose tissue is caused by PDE3, probably by the isoform PDE3B.

cUMP hydrolysis by PDE3A.

As previously reported, the cardiac phosphodiesterase PDE3A hydrolyzes cUMP. Moreover, cUMP-degrading activity was detected in cow and dog hearts several decades ago. Our aim was to characterize the enzyme kinetic parameters of PDE3A-mediated cUMP hydrolysis and to investigate whether cUMP and cUMP-hydrolyzing PDEs are present in cardiomyocytes. PDE3A-mediated cUMP hydrolysis was characterized in time course, inhibitor, and Michaelis-Menten kinetics experiments. Intracellular cyclic nucleotide (cNMP) concentrations and the mRNAs of cUMP-degrading PDEs were quantitated in neonatal rat cardiomyocytes (NRCMs) and murine HL-1 cardiomyogenic cells. Moreover, we investigated cUMP degradation in HL-1 cell homogenates and intact cells. Educts (cNMPs) and products (NMPs) of the PDE reactions were detected by HPLC-coupled tandem mass spectrometry. PDE3A degraded cUMP (measurement of UMP formation) with a K M value of ~143 µM and a V max value of ~42 µmol/min/mg. PDE3A hydrolyzed cAMP with a K M value of ~0.7 µM and a V max of ~1.2 µmol/min/mg (determination of AMP formation). The PDE3 inhibitor milrinone inhibited cUMP hydrolysis (determination of UMP formation) by PDE3A (K i = 57 nM). Significant amounts of cUMP as well as of PDE3A mRNA (in addition to PDE3B and PDE9A transcripts) were detected in HL-1 cells and NRCMs. Although HL-1 cell homogenates contain a milrinone-sensitive cUMP-hydrolyzing activity, intact HL-1 cells may use additional PDE3-independent mechanisms for cUMP disposal. PDE3A is a low-affinity and high-velocity PDE for cUMP. Future studies should investigate biological effects of cUMP in cardiomyocytes and the role of PDE3A in detoxifying high intracellular cUMP concentrations under pathophysiological conditions.

The histamine H4-receptor and the central and peripheral nervous system: A critical analysis of the literature.

Expression and function of histamine H4R in central and peripheral nervous system have been a matter of controversy for more than a decade. The scientific discussion is often limited to a few publications postulating the presence of functional H4R on neurons of the central and peripheral nervous system, but the even larger number of reports showing negative data is often neglected. In this article, we critically review the existing literature on H4R in central and peripheral nervous system and discuss the weak points often overlooked by the community. We identified as most important problems (i) insufficient validation or quality of antibodies, (ii) missing knockout controls, (iii) uncritical interpretation of RT-PCR results instead of qPCR experiments, (iv) insufficient controls to confirm specificity of pharmacological tools, (v) uncritical reliance on results produced by a single method and (vi) uncritical reliance on results not reproduced by independent research groups. Additionally, there may be a publication as well as a citation bias favoring the awareness of positive results, but neglecting negative data. We conclude that H4R expression on neurons of the brain is not convincingly supported by the current literature, at least as long as the positive data are not reproduced by independent research groups. Expression and function of H4R on peripheral neurons or non-neuronal cells of the nervous system, specifically on microglia is an interesting alternative hypothesis that, however, requires further verification. This article is part of a Special Issue entitled 'Histamine Receptors'.

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.

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.

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).