The Receptor-Bound Guanylyl Cyclase DAF-11 Is the Mediator of Hydrogen Peroxide-Induced cGMP Increase in Caenorhabditis elegans [corrected]..

Adenosine 3', 5'-cyclic monophosphate (cAMP) and guanosine 3', 5'-cyclic monophosphate (cGMP) are well-studied second messengers that transmit extracellular signals into mammalian cells, with conserved functions in various other species such as Caenorhabditis elegans (C. elegans). cAMP is generated by adenylyl cyclases, and cGMP is generated by guanylyl cyclases, respectively. Studies using C. elegans have revealed additional roles for cGMP signaling in lifespan extension. For example, mutants lacking the function of a specific receptor-bound guanylyl cyclase, DAF-11, have an increased life expectancy. While the daf-11 phenotype has been attributed to reductions in intracellular cGMP concentrations, the actual content of cyclic nucleotides has not been biochemically determined in this system. Similar assumptions were made in studies using phosphodiesterase loss-of-function mutants or using adenylyl cyclase overexpressing mutants. In the present study, cyclic nucleotide regulation in C. elegans was studied by establishing a special nematode protocol for the simultaneous detection and quantitation of cyclic nucleotides. We also examined the influence of reactive oxygen species (ROS) on cyclic nucleotide metabolism and lifespan in C. elegans using highly specific HPLC-coupled tandem mass-spectrometry and behavioral assays. Here, we show that the relation between cGMP and survival is more complex than previously appreciated.

Nucleotidyl cyclase activity of particulate guanylyl cyclase A: comparison with particulate guanylyl cyclases E and F, soluble guanylyl cyclase and bacterial adenylyl cyclases CyaA and edema factor.

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1ß1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.

Purine metabolism during neuronal differentiation: the relevance of purine synthesis and recycling.

Purines are a class of small organic molecules that are essential for all cells. They play critical roles in neuronal differentiation and function. Their importance is highlighted by several inherited disorders of purine metabolism, such as Lesch-Nyhan disease, which is caused by a deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). Despite the known importance of purines in the nervous system, knowledge regarding their metabolism in neurons is limited. In the current studies, purine pools and their metabolism were examined in rat PC6-3 cells, a PC12 pheochromocytoma subclone that undergoes robust differentiation with nerve growth factor. The results were compared with five new independent PC6-3 subclones with defective purine recycling because of different mutations affecting HGprt enzyme activity. The results demonstrate an increase in most purines and in energy state following neuronal differentiation, as well as specific abnormalities when purine recycling is lost. The loss of HGprt-mediated purine recycling also is associated with significant loss of dopamine and related metabolites in the mutant PC6-3 lines, suggesting an important connection between purine and dopamine pathways. These results provide insights into how purine pools and metabolism change with neuronal differentiation, and how specific enzyme defects may cause neuronal dysfunction. Differentiation of dopaminergic PC6-3 cells is accompanied by increased purine pools and energy state. The lack of a functional purine recycling pathway causes purine limitation in both undifferentiated and differentiated cells, as well as profound loss of dopamine content. The results imply an unknown mechanism by which intracellular purine levels regulate dopamine levels.

The dual H3/4R antagonist thioperamide does not fully mimic the effects of the 'standard' H4R antagonist JNJ 7777120 in experimental murine asthma.

Histamine is detected in high concentrations in the airways during an allergic asthma response. In a murine model of allergic asthma, the histamine H4 receptor (H4R)-selective ligand JNJ 7777120 reduces asthma-like symptoms. A sole antagonistic function of JNJ 7777120 at the murine H4R has, however, been questioned in the literature. Therefore, in the present study, we aimed at analyzing the effects of JNJ 7777120 in comparison to that of the H3/4R-selective antagonist thioperamide. Experimental murine asthma was induced by sensitization and provocation of BALB/c mice with ovalbumine (OVA). JNJ 7777120, thioperamide, or JNJ 5207852, an H3R-selective antagonist which was used to dissect H3R- and H4R-mediated activities of thioperamide, were injected subcutaneously during sensitization and effects were analyzed after provocation. Pharmacokinetic analyses revealed shortest t1/2 values in both plasma and lung tissue and lowest maximal concentration in lung tissue for JNJ 7777120 in comparison to thioperamide and JNJ 5207852. Nevertheless, JNJ 7777120 reduced serum titers of allergen-specific (anti-OVA) IgE, inflammatory infiltrations in lung tissue, and eosinophilia in bronchoalveolar lavage fluid. In contrast, thioperamide reduced only eosinophilia in bronchoalveolar lavage fluid, while anti-OVA IgE concentrations and lung infiltrations remained unaffected. JNJ 5207852 had no effect on these parameters. JNJ 7777120 provides beneficial effects in experimental murine asthma, which, however, could only partially be mimicked by thioperamide, despite more favorable pharmacokinetics. Thus, whether these effects of JNJ 7777120 are entirely attributable to an antagonistic activity at the murine H4R or whether an agonistic activity is also involved has to be reconsidered.

Dissociations in the effects of ß2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils: support for the concept of functional selectivity.

In neutrophils, activation of the ß2-adrenergic receptor (ß2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various ß2AR ligands on adenosine-3',5'-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2(•-)) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2(•-) formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. ß2AR agonists were generally more potent in inhibiting fMLP-induced O2(•-) production than in stimulating cAMP accumulation. (-)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2(•-) production. Moreover, (-)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2(•-) assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2(•-) production after ß2AR-stimulation although cAMP-increasing substances inhibited O2(•-) production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the ß2AR inhibits O2(•-) production in a cAMP-independent manner.

Quantification of cyclic dinucleotides by reversed-phase LC-MS/MS.

Cyclic dinucleotides such as bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) represent important second messengers in bacteria. Although their synthesis has not been described in plants so far, they may be involved in the regulation of bacterial phytopathogen-plant interactions as well as rhizobium plant symbiosis. Here, we describe a sensitive and specific quantification method for c-di-AMP and c-di-GMP by HPLC-coupled tandem mass spectrometry. Additional linear dinucleotide metabolites and mononucleotides, as well as cyclic mononucleotides, can be simultaneously determined by this method.

Impairment of adenylyl cyclase 2 function and expression in hypoxanthine phosphoribosyltransferase-deficient rat B103 neuroblastoma cells as model for Lesch-Nyhan disease: BODIPY-forskolin as pharmacological tool.

Hypoxanthine phosphoribosyl transferase (HPRT) deficiency results in Lesch-Nyhan disease (LND). The link between the HPRT defect and the self-injurious behavior in LND is still unknown. HPRT-deficient rat B103 neuroblastoma cells serve as a model system for LND. In B103 cell membranes, HPRT deficiency is associated with a decrease of basal and guanosine triphosphate-stimulated adenylyl cyclase (AC) activity (Pinto and Seifert, J Neurochem 96:454-459, 2006). Since recombinant AC2 possesses a high basal activity, we tested the hypothesis that AC2 function and expression is impaired in HPRT deficiency. We examined AC regulation in B103 cell membranes, cAMP accumulation in intact B103 cells, AC isoform expression, and performed morphological studies. As most important pharmacological tool, we used 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene forskolin (BODIPY-FS) that inhibits recombinant AC2 but activates ACs 1 and 5 (Erdorf et al., Biochem Pharmacol 82:1673-1681, 2011). In B103 control membranes, BODIPY-FS reduced catalysis, but in HPRT(-) membranes, BODIPY-FS was rather stimulatory. 2'(3')-O-(N-methylanthraniloyl) (MANT)-nucleoside 5'-[γ-thio]triphosphates inhibit recombinant ACs 1 and 5 more potently than AC2. In B103 control membranes, MANT-guanosine 5'-[γ-thio]triphosphate inhibited catalysis in control membranes less potently than in HPRT(-) membranes. Quantitative real-time PCR revealed that in HPRT deficiency, AC2 was virtually absent. In contrast, AC5 was up-regulated. Forskolin (FS) and BODIPY-FS induced cell clustering and rounding and neurite extension in B103 cells. The effects of FS and BODIPY-FS were much more prominent in control than in HPRT(-) cells, indicative for a differentiation defect in HPRT deficiency. Neither FS nor BODIPY-FS significantly changed cAMP concentrations in intact B103 cells. Collectively, our data show that HPRT deficiency in B103 cells is associated with impaired AC2 function and expression and reduced sensitivity to differentiation induced by FS and BODIPY-FS. We discuss the pathophysiological implications of our data for LND.

Pharmacokinetics of ampicillin/sulbactam in critically ill patients with acute kidney injury undergoing extended dialysis.

BACKGROUND AND OBJECTIVES: The fixed antibacterial combination of ampicillin and sulbactam is frequently used for various infections. Intact kidneys eliminate approximately 71% of ampicillin and 78% of sulbactam. Patients on thrice-weekly low-flux hemodialysis exhibit an ampicillin t(1/2) of 2.3 hours on and 17.4 hours off dialysis. Despite its frequent use in intensive care units, there are no available dosing recommendations for patients with AKI undergoing renal replacement therapy. The aims of this study were to evaluate the pharmacokinetics of ampicillin/sulbactam in critically ill patients with AKI undergoing extended dialysis (ED) and to establish a dosing recommendation for this treatment method. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Twelve critically ill patients with anuric AKI being treated with ED were enrolled in a prospective, open-label, observational pharmacokinetic study. Pharmacokinetics after a single dose of ampicillin/sulbactam (2 g/1 g) was obtained in 12 patients. Multiple-dose pharmacokinetics after 4 days of twice-daily ampicillin/sulbactam (2 g/1 g) was obtained in three patients. RESULTS: The mean dialyzer clearance for ampicillin/sulbactam was 80.1 ± 7.7/83.3 ± 12.1 ml/min. The t(1/2) of ampicillin and sulbactam in patients with AKI undergoing ED were 2.8 ± 0.8 hours and 3.5 ± 1.5 hours, respectively. There was no significant accumulation using a twice-daily dosage of 2 g/1 g ampicillin/sulbactam. CONCLUSIONS: Our data suggest that in patients treated with ED using a high-flux dialyzer (polysulphone, 1.3 m(2); blood and dialysate flow, 160 ml/min; treatment time, 480 minutes), a twice-daily dosing schedule of at least 2 g/1 g ampicillin/sulbactam, with one dose given after ED, should be used to avoid underdosing.

Quantitative analysis of the antifungal drug anidulafungin by LC-online SPE-MS/MS in human plasma.

The objective of this study was to develop a fast and robust method for the quantitation of the antifungal drug anidulafungin in human plasma samples by generic two-dimensional liquid chromatography (online-SPE/reversed phase LC) coupled to a tandem-quadrupole mass spectrometer (LC-online SPE-MS/MS). Online SPE was performed using an Oasis HLB cartridge column and for reversed-phase chromatography a Nucleodur Gravity C(18) column was used. A 100 µL aliquot of human plasma was extracted with 200 µL of 80:20 MeOH-0.2 M ZnSO(4) (v/v) as precipitation reagent containing ascomycin as internal standard (IS). The supernatant was directly injected for analysis. The total run time was 4.5 min. Anidulafungin and ascomycin were detected in the positive ionization mode. The method performance data for anidulafungin, such as limit of detection (0.013 µg/mL), lower limit of quantitation (0.04 µg/mL), linearity (R(2) = 0.9999) and concentration range (0.04-10 µg/mL) were ascertained. Intra- and inter-day precisions were ≤6.6% and intra- and inter-day accuracies were 98.5-101.0 and 100.0-102.5%, respectively. The assay was successfully applied for quantitation of anidulafungin in patient plasma samples.

Nucleotidyl cyclase activity of soluble guanylyl cyclase α1ß1.

Soluble guanylyl cyclase (sGC) regulates several important physiological processes by converting GTP into the second-messenger cGMP. sGC has several structural and functional properties in common with adenylyl cyclases (ACs). Recently, we reported that membranous ACs and sGC are potently inhibited by 2',3'-O-(2,4,6-trinitrophenyl)-substituted purine and pyrimidine nucleoside 5'-triphosphates. Using a highly sensitive high-performance liquid chromatography-tandem mass spectrometry method, we report that highly purified recombinant sGC of rat possesses nucleotidyl cyclase activity. As opposed to GTP, ITP, XTP and ATP, the pyrimidine nucleotides UTP and CTP were found to be sGC substrates in the presence of Mn(2+). When Mg(2+) is used, sGC generates cGMP, cAMP, cIMP, and cXMP. In conclusion, soluble "guanylyl" cyclase possesses much broader substrate specificity than previously assumed. Our data have important implications for cyclic nucleotide-mediated signal transduction.

Human cyclic nucleotide phosphodiesterases possess a much broader substrate-specificity than previously appreciated.

Phosphodiesterases (PDEs) capable of degrading cAMP and cGMP are indispensable for the regulation of cyclic nucleotide-mediated signals. The existence of other cyclic nucleotides such as cCMP and cUMP has been discussed controversially in the literature. Despite publications on PDEs hydrolyzing cCMP or cUMP, the molecular identity of such enzymes remained elusive. Recently, we have provided evidence for a role of cCMP as second messenger in vascular relaxation and inhibition of platelet aggregation. Using an HPLC-MS based assay, here, we show that human PDEs belonging to various families hydrolyze not only cAMP and cGMP but also other cyclic nucleotides.

Systematic analysis of histamine and N-methylhistamine concentrations in organs from two common laboratory mouse strains: C57Bl/6 and Balb/c.

OBJECTIVE: Histamine plays a role in several (patho) physiological processes that are commonly studied in mouse models. However, a systematic quantification of histamine and its metabolite N-methylhistamine in mouse organs has not been reported so far. METHODS: Balb/c and C57Bl/6 mice were grouped according to their sex and age. Brains, hearts, lungs, livers, kidneys, stomachs, intestines, thymi, spleens, and lymph nodes were excised, weighed, and homogenized. Histamine and N-methylhistamine were quantified simultaneously by a HPLC-mass spectrometry method. RESULTS: In all organs analyzed, histamine and N-methylhistamine were detected; however, with quantitative differences. Histamine was present most abundantly in the stomach, lymph nodes, and thymus. The lowest histamine concentrations were detected in brain, liver, lung, and intestine. In most organs, the histamine concentrations increased age-dependently. Substantial concentrations of N-methylhistamine were detected only in lung, intestine and kidney, while in all other organs it was present only in minor quantities. CONCLUSION: HPLC-mass spectrometry is a useful method for the highly sensitive and simultaneous detection of histamine and N-methylhistamine. Histamine is present in virtually all organs, not only in those traditionally associated with histamine-mediated disease. Highest concentrations are found in stomach, lymph node, and thymus; medium concentrations in heart, spleen, and kidney; and lowest concentrations detected in intestine, brain, liver, and lung.

c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress.

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13-22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.

Risk of underdosing of ampicillin/sulbactam in patients with acute kidney injury undergoing extended daily dialysis--a single case.

The fixed antibacterial combination of ampicillin and sulbactam is frequently used for various infections. The normal kidneys eliminate approximately 60% of ampicillin (371.39 Da) and sulbactam (255.22 Da). Concomitant with the decline in renal function, the terminal elimination half-life increases from 1 up to 24 h in patients with ESRD. Patients on three times weekly low flux haemodialysis exhibit a half-life of 2.3 h on and 17.4 h off dialysis. In contrast, in the present observation the elimination half-life in a single patient with acute kidney injury undergoing extended daily dialysis (EDD) with a polysulphone membrane was 1.5 h, indicating that the current dosing regimen for haemodialysis outpatients (ampicillin/sulbactam 2.0/1.0 g/day) would result in a significant underdosing for patients undergoing EDD.

Quantification of antiretroviral drugs in dried blood spot samples by means of liquid chromatography/tandem mass spectrometry.

For the first time approved antiretroviral drugs, i.e. protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI), were quantified in dried blood spots (DBS) from HIV/AIDS patient whole blood samples as the basis for therapeutic drug monitoring (TDM) by a robust simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) method. This study included seven PI (amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, atazanavir) and two NNRTI (nevirapine, efavirenz). LC/MS/MS coupling was realized using a Phenomenex Synergy Max RP LC column (150 x 2 mm, 4 micro) in combination with a tandem mass spectrometer (API 2000, Applied Biosystems/MDS Sciex Concord) operating in positive and negative multiple reaction monitoring (MRM) mode with reserpine as internal standard. DBS samples were punched out and extracted with 50:50 MeOH/0.2 M ZnSO4 (v/v) as extraction reagent. The method performance data for the drugs in DBS like limits of detection (LOD, 8-70 ng/mL), lower limits of quantification (LLOQ, 41-102 ng/mL), linearity (R2, 0.9981-0.9999), linear concentration ranges (41-10.000 ng/mL), accuracies (92-113%), recoveries (62-94%), and ion suppression were investigated and are comparable to data obtained from human plasma, which is the current standard matrix for TDM of PI and NNRTI. In this case, off-line plasma sample preparation was performed by means of simple protein precipitation with 80:20 methanol/0.2 M ZnSO4 (v/v) as precipitation reagent. Significant correlations between real patient plasma and DBS were obtained for samples containing lopinavir, atazanavir, ritonavir, saquinavir, and efavirenz. DBS preparation as sampling alternative is well suited and practicable for TDM minimizing the high infection risk of HIV/AIDS samples and may facilitate sample mailing.