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Root exudation is a major pathway of organic carbon input into soils. It affects soil physical properties, element solubility as well as speciation, and impacts the microbial community in the rhizosphere. Root exudates contain a large number of primary and secondary plant metabolites, and the amount and composition are highly variable depending on plant species and developmental stage. Detailed information about exudate composition will allow for a better understanding of exudate-driven rhizosphere processes and their feedback loops. Although non-targeted metabolomics by high-resolution mass spectrometry is an established tool to characterize root exudate composition, the extent and depth of the information obtained depends strongly on the analytical approach applied. Here, two genotypes of Zea mays L., differing in root hair development, were used to compare six mass spectrometric approaches for the analysis of root exudates. Reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography combined with time-of-flight mass spectrometry (LC-TOF-MS), as well as direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS), were applied with positive and negative ionization mode. By using the same statistical workflow, the six approaches resulted in different numbers of detected molecular features, ranging from 176 to 889, with a fraction of 48 to 69% of significant features (fold change between the two genotypes of > 2 and p-value rth3). These results were in agreement with the higher total carbon and nitrogen exudation rate of the rth3-mutant as compared to the corresponding wild-type maize (WT). However, only a small fraction of features were commonly found across the different analytical approaches (20–79 features, 13–31% of the rth3-mutant up-regulated molecular formulas), highlighting the need for different mass spectrometric approaches to obtain a more comprehensive view into the composition of root exudates. In summary, 111 rth3-mutant up-regulated compounds (92 different molecular formulas) were detected with at least two different analytical approaches, while no WT up-regulated compound was found by both, LC-TOF-MS and DI-FT-ICR-MS. Zea mays L. exudate features obtained with multiple analytical approaches in our study were matched against the metabolome database of Zea mays L. (KEGG) and revealed 49 putative metabolites based on their molecular formula. Graphical Abstract

Detection of DBPs directly from only 1 μL of drinking water provides novel and less biased insights of DBP formation and their chemical properties.

Mendelian syndromes give great insight into pathogenesis and have implicated salt handling. Hypertension with brachydactyly (HTNB) is unique in that a direct increase in peripheral vascular resistance is produced by activating mutations in phosphodiesterase 3A (PDE3A). A 50 mm Hg blood-pressure elevation by age 50 years causes stroke in untreated persons. Here, we report mutations in the PDE3 catalytic domain found in two new HTNB families. Since structural predictions indicated increased PDE3A cleavage activity, we used Förster resonance energy transfer (FRET) to measure the PDE3-specific cAMP degradation in cytosolic fractions from transfected cells. The newly discovered catalytic-domain PDE3A mutants, R862C and L910P, both showed a shorter transient emission-ratio change upon cAMP addition than PDE3A wildtype, indicating faster cAMP turnover. The V max of all experiments were extracted from FRET data and quantitated and analyzed statistically. The mean values V max (nM (cAMP)/s) were 143±7 for L910P, 63±3 for R862C and 73±4 for T445N, and 45±3 for WT respectively, (p

The SARS-CoV-2 pandemic has shown the importance of rapid and comprehensive diagnostic tools. While there are numerous rapid antigen tests available, rapid serological assays for the detection of neutralizing antibodies are and will be needed to determine not only the amount of antibodies formed after infection or vaccination but also their neutralizing potential, preventing the cell entry of SARS-CoV-2. Current active-virus neutralization assays require biosafety level 3 facilities, while virus-free surrogate assays are more versatile in applications, but still take typically several hours until results are available. To overcome these disadvantages, we developed a competitive chemiluminescence immunoassay that enables the detection of neutralizing SARS-CoV-2 antibodies within 7 min. The neutralizing antibodies bind to the viral receptor binding domain (RBD) and inhibit the binding to the human angiotensin-converting enzyme 2 (ACE2) receptor. This competitive binding inhibition test was characterized with a set of 80 samples, which could all be classified correctly. The assay results favorably compare to those obtained with a more time-intensive ELISA-based neutralization test and a commercial surrogate neutralization assay. Our test could further be used to detect individuals with a high total IgG antibody titer, but only a low neutralizing titer, as well as for monitoring neutralizing antibodies after vaccinations. This effective performance in SARS-CoV-2 seromonitoring delineates the potential for the test to be adapted to other diseases in the future. Graphical Abstract

The in vivo quantification of metabolic products from microbial single cells is one of the last grand challenges in (bio)analytical chemistry. To date, no label-free analytical concept exists that is powerful enough to detect or even quantify the minute amounts of secreted low molecular weight compounds produced by living and isolated single bacteria or yeast cells. Coupling microfluidic cultivation systems with ultrahigh resolution electrospray-ionization mass spectrometry with its exquisite sensitivity and specificity offers the prospect of single-cell product analysis and quantification, but has not been successfully implemented yet. We report an analytical framework that interfaces noninvasive microfluidic trapping and cultivation of a few bacterial single cells with the analysis of their catalytic products by Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Cell trapping was performed with the microfluidic Envirostat platform for cultivating bacterial cells under continuous perfusion via negative dielectrophoresis (nDEP). A total of 1.5 μL of product-containing cell supernatant was sampled into microcapillaries using a dead volume-reduced world-to-chip interface. The samples were analyzed with a nanoESI ion source coupled to a FT-ICR-MS (limit of detection for lysine: 0.5 pg). As a biocatalytic model system, we analyzed few Corynebacterium glutamicum DM 1919 pSenLys cells that synthesized l-lysine from d-glucose. Secreted lysine was quantified from a few cells (down to 19). Single-cell specific lysine productivities were 2 and 10 fmol/cell/h. This demonstrates that coupling microfluidics and mass spectrometry (SIC-MS) now enables the quantification of catalytic products and extracellular metabolites from only a few living microbial cells.

The rhizosphere is an important hotspot for microbial activity, organic carbon input, and carbon turnover in soils. The interplay of these rhizosphere components results in small scale gradients of organic molecules in the zone around a root. Mass spectrometric imaging (MSI) can reveal the spatial distribution of individual plant metabolites in the soil, which cannot be achieved using bulk analysis. Using non-fragmenting ionization techniques such as laser desorption ionization (LDI) allows for the detection of intact molecules without the need for labeling with e.g. fluorescent tags.Direct MSI for the chemical imaging of intact molecules of the rhizosphere has been recognized as a still existing analytical gap. Here we present a novel method allowing mass spectrometric molecular rhizosphere imaging directly in a complex soil matrix.Our novel approach consists of sampling the roots and the surrounding soil of Zea mays plants in either field- or lab-scale experiments using small metal cylinders. After excavation, the loam soil pellets were embedded in gelatin and cryosectioned to 100 µm sections. After selecting regions of interest on the soil section, the root and the soil surrounding the root was analysed using ultra-high resolution laser desorption ionization Fourier-transform ion cyclotron resonance mass spectrometry (LDI-FT-ICR-MS).Given the large background of soil-derived organic carbon, the high mass resolution and sensitivity of FT-ICR-MS allow distinguishing root-derived molecules from soil organic matter based on their exact masses. We show that our method is capable to recover rhizosphere gradients of a dihexose (C12H22O11, e.g. sucrose, maltose) directly in the soil with a spatial resolution of 25 µm.Molecular gradients for the dihexose showed a high abundance of this metabolite in the root and a strong depletion of the signal intensity within 150 µm from the root surface. Analysing several sections from the same soil pellet allowed to recover 3D molecular gradients from one root segment. Utilizing the potential to easily change the mass window a variety of potential metabolites can be analysed in the same region around the root. Thus the chemical diversity of potential root exudates can be revealed.Our workflow enables the study of root-derived organic carbon with high spatial resolution directly in a soil context. For the first time, direct molecular imaging of the rhizosphere via LDI-FT-ICR-MS will allow for a non-target or targeted analysis of complex soil samples.Visualizing the root structure via X-ray computed tomography in a soil sample before the embedding would enable a guided sampling approach to analyse molecular distributions at certain parts of the root. Moreover, the molecular LDI-MSI results could be correlated with elemental imaging via laser ablation – inductively coupled plasma – mass spectrometry directly at the same sample position - allowing for an even more detailed insight into chemical processes in the rhizosphere.

„Podiumsdiskussion: Was lernen wir aus der Vergangenheit, wie entwickeln wir Impfstoffe, Impfstrategien und Therapien fur die Zukunft?“

A new method combining online nano solid phase extraction coupled with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was developed to extract and analyze organic matter (OM) from microliter volumes of salt containing soil solution samples. This approach allows the reproducible analysis of only minute amounts of organic carbon (down to 10 ng C) without the need of further sample preparation. The new method was applied to unravel developing small-scale patterns of dissolved organic matter (DOM) in soil solutions of a soil column experiment in which Zea mays plants were grown for 3 weeks. Soil solution was sampled by micro suction cups from the undisturbed soil-root system once a week. Growth of the root system and, hence, position of individual roots relative to the suction cups was followed by X-ray computed tomography (X-ray CT). Our method makes it possible to resolve the chemical complexity of soil solution OM (up to 4300 molecular formulas from 2.5 μL sample). This allows to observe chemical gradients in the rhizosphere on a molecular level over time. The increasing influence of roots on soil solution OM is visible from higher molecular masses, an increasing degree of oxygenation and a higher fraction of formulas containing heteroatoms. The online nano solid phase extraction-FT-ICR-MS method provides novel insight into the processes affecting DOM in the rhizosphere, such as root exudation, microbial processes, and soil organic matter stabilization.

Plant-microbe interplay in the rhizosphere generates multi-faceted chemical gradients. The soil solution is a crucial component of the rhizosphere, where chemical gradients of organic molecules first develop upon growth of roots, introduction of plant-derived carbon and microbial turnover. Studying these gradients requires high resolution both in time and space as well as high chemical specificity to resolve the multitude of compounds. Existing methods to probe the rhizosphere soil solution were mostly limited to bulk chemical parameters, inorganic ions or targeted analysis of organic molecules. However, to decipher organic carbon turnover in the rhizosphere the characterization of the complex pool of soil solution organic matter is needed.Here we present a novel method that combines time-resolved collection of soil solution samples via micro-suction cups in the rhizosphere with ultrahigh chemical resolution provided by Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to unravel developing pattern of soil solution organic matter.Zea mays plants were grown in soil columns for three weeks and soil solution samples of undisturbed root-soil system were collected once a week. Growth of the root system and hence position of sampling locations in relation to the distance from the root, were followed by X-ray computed tomography (X-ray CT).The online sample preparation was optimised to extract and desalt the organic matter from a few microliters of soil solution. The downscaling to a nano-liquid chromatography system for the on-line extraction allowed the analysis of only minute amounts of organic carbon within the samples (down to 10 ng). Given the high background concentration of soil-derived organic carbon, the high mass resolution and sensitivity of FT-ICR-MS enabled to distinguish root derived molecules from soil organic matter based on their exact masses. Molecular formulas of the root derived molecules could be calculated showing distinct chemical characteristics as compared to the bulk soil solution. X-ray CT analyses enabled relating the results from the chemical analysis to distance from the root and root age. With increasing influence of the roots higher molecular masses and an increasing degree of oxygenation of the molecules could be observed.Our method is thus capable to show the changing small scale pattern of soil solution organic matter during the early rhizosphere development, closing the knowledge gap between root exudates, soil chemistry and microbial processes.

Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of Ne-acryloyllysine piperazides as irreversible inhibitors of TGase 2. Systematic structural modifications on 54 new compounds were performed with a major focus on fluorine-bearing substituents due to the potential of such compounds to serve as radiotracer candidates for positron emission tomography. The determined inhibitory activities ranged from 100 to 10 000 M–1 s–1, which resulted in comprehensive structure–activity relationships. Structure–activity correlations using various substituent parameters accompanied by covalent docking studies provide an advanced understanding of the...

To date, several numerical models predicting the properties of TIG-arcs are available. Recently, effort has been put into testing the reliability of these models for varying process parameters by means of comparing plasma temperatures and arc voltages against measured data. However, from an engineering point of view, the goal of these models is to predict the properties of weld beads. Therefore, the heat input into and the pressure applied onto the workpiece have to be predicted. This paper deals with the comparison of measured arc pressure data and the results of an approach which couples a simplified cathode sheath model with an LTE arc model.

Laser-based joining is a potential key manufacturing process for realizing metal plastic hybrids. Therefore, surface preparation is essential to achieve a mechanical form fit. A new approach for surface preparation is based on a tungsten inert gas (TIG) arc process with anodic polarity. This TIG structure was characterized compared to fiber laser manufactured structures in cw and pw mode. The comparison of different preparations was based on pure ultimate tensile strength tests on spot joints. Finally, a transfer to overlap joints was successfully carried out and characterized by tensile shear tests using fiber reinforced plastics.

Within the framework of the bilateral CORNET projects MeTexCom and MeTexCom2, new approaches were developed and tested to improve the adhesion strength of metal textile composites, with a focus on the targeted roughening of aluminum surfaces and the development of new acoustically insulating nonwovens. The metal textile composites were produced by melting thermoplastic components of the textile composites without a separately applied adhesive.For improved adhesion strength between metal and textile, roughness was generated on the metal surface by means of a novel arc treatment by an anodic polarized TIG process or a cw (continuous wave) fiber laser process. On the one hand, the goal was to produce uniformly rough, untercut surface structures in micro-and nanodimension by means of a highly dynamic arcing process. On the other hand, a similar approach was pursued with the cw laser method by using a single-mode as well as a multi-mode laser.

Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of N

To date, several numerical models representing the tungsten inert gas (TIG) arc are available. However, little has been done on testing the reliability of these models for parameter values differing from the ones used in the respective papers. This paper deals with the comparison of measured arc welding data provided by E. Siewert from Linde AG and the results of the two most common arc and cathode sheath models. These models make different degrees of simplification of the actual processes in the arc, especially the cathode sheath. Different shapes of the electrodes, different welding currents and shielding gases are taken into account as well as the resolution of the numerical grid on which the calculations are carried out. The findings of this paper are used to show when a simplified approach still leads to valid results and when more sophisticated models have to be used.

In this paper, the results for keyhole welding of stainless steel 1.4301 and mild steel 1.0117 with a cathode focussed gas tungsten arc process are presented. These tests were carried out in preparation for welding the root faces of Y- or double V-groove weld preparations of thick metal plates. Welded plate thicknesses range from 6 to 10 mm for stainless steels and 6 to 12 mm for mild steels. Welding speeds are in the range of 20 to 115 cm/min at currents from 400 to 800 A. Especially for stainless steel, a big range of applicable and stable process parameters could be found. The process also works fine for mild steel even though the achievable parameter range is smaller.

This paper presents extensive investigations about the influence of He-additions to argon TIG-arc properties. Results of diagnostic methods, numerical simulations and welding trials are combined to improve the comprehension about the mode of action of gas mixtures. Additionally, selected results of the investigation with H2- and N2-additions are summarized. The investigations show that all gas additions cause an increase of the heat input into the workpiece. However, the stagnation pressure depends on the gas composition: He-additions result in a decrease of the stagnation pressure which depends on the arc length, whereas H2-and N2-additions increase the pressure. By a systematic choice of the gas mixture the weld depth and also the maximum feed speed can be influenced.

Achievable benefits in hybrid laser-arc welding are closely related to suitable parameter settings. Basic optimizations consequently need a profound understanding of the relevance of involved interaction mechanisms. These are however differently evaluated and discussed in literature. This paper gives an over-view on the most popular hypotheses in the field of laser-arc processing. The importance of direct interactions between laser radiation and arc plasma as well as the role of metal evaporation are particularly considered by own experimental and numerical investigations. The results of this analysis suggest that these phenomena cannot be regarded as main driving mechanisms for synergistic achievements in hybrid laser-arc welding with respect to process stability and performance.Achievable benefits in hybrid laser-arc welding are closely related to suitable parameter settings. Basic optimizations consequently need a profound understanding of the relevance of involved interaction mechanisms. These are however differently evaluated and discussed in literature. This paper gives an over-view on the most popular hypotheses in the field of laser-arc processing. The importance of direct interactions between laser radiation and arc plasma as well as the role of metal evaporation are particularly considered by own experimental and numerical investigations. The results of this analysis suggest that these phenomena cannot be regarded as main driving mechanisms for synergistic achievements in hybrid laser-arc welding with respect to process stability and performance.

G protein-coupled receptor kinases (GRKs) belong to the AGC family of serine/threonine kinases. They phosphorylate agonist-activated G protein-coupled receptors (hence the name). This phosphorylation triggers the binding of arrestins to the receptors. Receptor:arrestin interaction then leads to the desensitization of G protein activation by the receptors and can also lead to internalization of the receptors and initiation of new signaling pathways. Some G protein-coupled receptor kinases can also constitutively phosphorylate cytosolic proteins, including beta-tubulin …

We tested the hypothesis that the human beta(1)-adrenergic receptor displays constitutive activity and that beta-adrenergic antagonists differ in their ability to modulate this constitutive activity. Transfection of the cDNAs of the human beta(1)- and beta(2)-adrenergic receptors into COS-7 cells caused increases in basal cAMP that were proportional to the receptor levels, thus demonstrating constitutive activity for both subtypes. At comparable receptor levels, the increase in basal cAMP was about 5-fold higher for the beta(2)- than for the beta(1)-subtype. As a model for enhanced beta-adrenergic signaling at the whole-organ level, we used transgenic mice with heart-specific overexpression of the human beta(1)-adrenergic receptor. In this model, the beta(1)-adrenergic receptor displayed constitutive activity as evidenced by a higher spontaneous beating rate of isolated right atria from beta(1)-transgenic versus wild-type mice. This difference was abolished by the addition of CGP20712A, demonstrating inverse agonist properties of this compound. We then tested whether various beta-adrenergic antagonists currently in clinical use for the treatment of heart failure differ in their ability to modulate constitutive activity of the cardiac beta(1)-adrenergic receptor. The beta(1)-selective antagonists metoprolol and bisoprolol showed significant inverse agonist activity at the beta(1)-adrenergic receptor. Carvedilol behaved as a neutral antagonist and xamoterol displayed marked partial agonist activity. We conclude that the human beta(1)-adrenergic receptor displays constitutive activity that is considerably lower than that of the beta(2)-subtype. beta-Adrenergic antagonists currently in clinical use differ in their ability to exert inverse agonist activity at the human beta(1)-adrenergic receptor, which may contribute to their therapeutic effects.

The paper presents a transient three-dimensional model of an anti-phase-synchronized pulsed tandem gas–metal arc welding process, which is used to analyse arc interactions and their influence on the gas shield flow. The shielding gases considered are pure argon and a mixture of argon with 18% CO2. Comparison of the temperature fields predicted by the model with high-speed images indicates that the essential features of the interactions between the arcs are captured. The paper demonstrates strong arc deflection and kinking, especially during the low-current phase of the pulse, in agreement with experimental observations. These effects are more distinct for the argon mixture with 18% CO2. The second part of the paper demonstrates the effects of arc deflection and instabilities on the shielding gas flow and the occurrence of air contamination in the process region. The results allow an improved understanding of the causes of periodic instabilities and weld seam imperfections such as porosity, spatter, heat-tint oxidation and fume deposits.

Receptor-specific or homologous desensitization of beta 2-adrenergic receptors is thought to be effected via phosphorylation of the receptor by the beta-adrenergic receptor kinase (beta ARK), followed by binding of beta-arrestin. We have generated stably transfected Chinese hamster ovary cell lines overexpressing either of the two regulatory proteins and also expressing low or high levels of beta 2-adrenergic receptors (approximately 80 and approximately 600 fmol/mg of membrane protein). In these cells, we studied the process of desensitization induced by the beta-adrenergic receptor agonist isoproterenol. In cells expressing high levels of beta 2-adrenergic receptors, desensitization to high concentrations of isoproterenol (previously shown to be mediated by both beta ARK and protein kinase A) amounted to approximately 50% in control cells, approximately 80% in beta ARK-overexpressing cells, and approximately 90% in beta-arrestin-overexpressing cells. In cells expressing low levels of beta 2-adrenergic receptors, these values were approximately 50, approximately 60, and approximately 60%, respectively. Desensitization to low concentrations of isoproterenol (previously shown to be essentially protein kinase A-mediated and not receptor-specific, i.e. heterologous) was not affected by overexpression of either beta ARK or beta-arrestin. These data suggest that in cells expressing high levels of beta 2-adrenergic receptors, beta-arrestin and beta ARK become limiting for homologous receptor desensitization. They provide further support for the involvement of these two proteins in the regulation of beta 2-adrenergic receptor function.

Homologous desensitization of beta-adrenergic receptors, as well as adaptation of rhodopsin, are thought to be triggered by specific phosphorylation of the receptor proteins. However, phosphorylation alone seems insufficient to inhibit receptor function, and it has been proposed that the inhibition is mediated, following receptor phosphorylation, by the additional proteins beta-arrestin in the case of beta-adrenergic receptors and arrestin in the case of rhodopsin. In order to test this hypothesis with isolated proteins, beta-arrestin and arrestin were produced by transient overexpression of their cDNAs in COS7 cells and purified to apparent homogeneity. Their functional effects were assessed in reconstituted receptor/G protein systems using either beta 2-adrenergic receptors with Gs or rhodopsin with Gt. Prior to the assays, beta 2-receptors and rhodopsin were phosphorylated by their specific kinases beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, respectively. beta-Arrestin was a potent inhibitor of the function of beta ARK-phosphorylated beta 2-receptors. Half-maximal inhibition occurred at a beta-arrestin:beta 2-receptor stoichiometry of about 1:1. More than 100-fold higher concentrations of arrestin were required to inhibit beta 2-receptor function. Conversely, arrestin caused half-maximal inhibition of the function of rhodopsin kinase-phosphorylated rhodopsin when present in concentrations about equal to those of rhodopsin, whereas beta-arrestin at 100-fold higher concentrations had little inhibitory effect. The potency of beta-arrestin in inhibiting beta 2-receptor function was increased over 10-fold following phosphorylation of the receptors by beta ARK, but was not affected by receptor phosphorylation using protein kinase A. This suggests that beta-arrestin plays a role in beta ARK-mediated homologous, but not in protein kinase A-mediated heterologous desensitization of beta-adrenergic receptors. It is concluded that even though arrestin and beta-arrestin are similar proteins, they display marked specificity for their respective receptors and that phosphorylation of the receptors by the receptor-specific kinases serves to permit the inhibitory effects of the "arresting" proteins by allowing them to bind to the receptors and thereby inhibit their signaling properties. Furthermore, it is shown that this mechanism of receptor inhibition can be reproduced with isolated purified proteins.

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied form of the beta-adrenergic and related G protein-coupled receptors. Structural features of this enzyme have been elucidated recently by the isolation of a cDNA that encodes bovine beta ARK. Utilizing a catalytic domain fragment of the beta ARK cDNA to screen a bovine brain cDNA library we have isolated a clone encoding a beta ARK-related enzyme which we have termed beta ARK2. Overall, this enzyme has 85% amino acid identity with beta ARK, with the protein kinase catalytic domain having 95% identity. The ability of beta ARK2 to phosphorylate various substrates was studied after expression in COS 7 cells. Although beta ARK2 is essentially equiactive with beta ARK in phosphorylating an acid-rich synthetic model peptide it was only approximately 50% as active when the substrate was the agonist-occupied beta 2-adrenergic receptor and only approximately 20% as active toward light-bleached rhodopsin. As with beta ARK, phosphorylation of the receptor substrates by beta ARK2 was completely stimulus dependent. RNA blot analysis with selected bovine tissues reveals an mRNA of 8 kilobases with a distribution similar to that of beta ARK. More detailed RNA analysis using a ribonuclease protection assay in various rat tissues suggests that the beta ARK2 message is present at much lower levels (typically 10-20%) than the beta ARK message. In the rat the beta ARK2 mRNA is localized predominantly in neuronal tissues although low levels are also observed in various peripheral tissues. The beta ARK2 gene has been localized to a region of mouse chromosome 5 whereas the beta ARK gene is localized on mouse chromosome 19. These data suggest the existence of a "family" of receptor kinases which may serve broadly to regulate receptor function.

It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine [(R)-AHPIA] into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels [Mol. Pharmacol. 30:403-409 (1986)]. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling, which is described in the accompanying paper [Mol. Pharmacol. 39:524-530 (1991)].

This study describes experiments investigating the mechanism of activation of A1 adenosine receptors. Isolated rat fat cells were used as a cellular model. The A1 receptors of these cells were covalently labeled with the agonist photoaffinity label R-2-azido-N6-p-hydroxyphenylisopropyladenosine. The covalent incorporation of the label into the binding subunit of the receptor was verified by demonstration of specific labeling of a peptide with Mr = 35,000 by the radioiodinated label. Such covalent labeling followed by removal of label not covalently bound led to a concentration-dependent reduction of cellular cAMP levels. This persistent effect of covalent labeling occurred with an IC50 value of 9 nM compared to an IC50 value of 0.9 nM for the direct reduction of cAMP levels by the label. The affinity of the label was determined in binding experiments. The Ki value of 19 nM was about 20 times higher than the corresponding IC50 value of cAMP reduction. Finally, the comparison between covalent binding and its effects suggests that covalently labeled receptors were fully activated. The data are interpreted as evidence for a receptor activation according to the occupancy theory. The analysis of the various concentration-response curves reveals the presence of spare receptors, which can be demonstrated by the method of agonist photoaffinity labeling.

Stimulation of beta 2-adrenergic receptors in intact cells causes, first, rapid functional uncoupling from Gs, which is triggered by receptor phosphorylation, and, second, somewhat slower sequestration of the receptors to an internal compartment. The present study addresses a possible role of sequestration in the resensitization of desensitized beta 2-adrenergic receptors in human A431 cells. Exposure of these cells to isoproterenol caused rapid phosphorylation, desensitization (as assessed in adenylyl cyclase assays), and sequestration of the receptors. Subsequent removal of the agonist led to recycling of the receptors to the cell surface, dephosphorylation, and restoration of receptor function. These effects occurred without any change in the total receptor number. The rate constant of agonist-induced sequestration was 0.03/min; the rate constant of receptor recycling was 0.06/min and was not markedly altered by the presence of agonist. Blockade of sequestration with concanavalin A or 0.6 M sucrose prevented receptor dephosphorylation as well as receptor resensitization. Inhibition of protein phosphatases with calyculin A caused a similar blockade of beta 2-adrenergic receptor resensitization; the effects of maximally effective concentrations of concanavalin A and calyculin A were not additive. Monensin impaired recycling of desensitized beta 2-adrenergic receptors to the cell surface and also prevented receptor resensitization. We conclude that sequestration of beta 2-adrenergic receptors, followed by dephosphorylation and recycling to the cell surface, may serve to restore the function of desensitized receptors.

Binding of the catecholamine agonists epinephrine and norepinephrine to the beta-adrenergic receptor (BAR) rapidly activates adenylate cyclase via the stimulatory guanine nucleotide regulatory protein Gs, and results in rises in cellular levels of cAMP. However, continuous exposure to these agonists leads within minutes to a dampening of the enzymatic response. Both in vivo and in vitro studies have implicated agonist-induced phosphorylation of BAR in this process. These results include the isolation of a novel beta-adrenergic receptor kinase (BARK), which has been shown to preferentially phosphorylate receptors that are occupied by agonist when assessed in vitro. Recent studies in our laboratory have examined the desensitization process in intact cells to determine where on the receptor molecule functionally relevant phosphorylation occurs, and to identify the kinase(s) involved. In one set of studies, site-specific mutagenic techniques with the cloned gene for the human beta 2-adrenergic receptor were utilized to delete putative sites of phosphorylation by BARK and/or the cAMP-dependent protein kinase (PKA). Following expression of the mutated receptors in mammalian cells, the cells were challenged with different concentrations of agonist for 10-15 min and the functional and phosphorylation properties of the mutant receptors were then assessed. In another set of studies human A431 cells were permeabilized with low concentrations of digitonin and treated with selective inhibitors of both BARK and PKA. The cells were then exposed to desensitizing concentrations of agonist, and similar measurements performed. Taken together, the results from both sets of studies suggest that exposure of cells to low (nanomolar) concentrations of agonist leads to phosphorylation of the receptor on one or both consensus sites for PKA, and that the predominant effect of this phosphorylation on the adenylyl cyclase response is a loss in sensitivity of the receptor to further stimulation by the agonist. In contrast, exposure of cells to higher (micromolar) concentrations of agonist leads to BAR phosphorylation by both PKA and BARK, the latter on the carboxyl terminal region of the receptor. Phosphorylation of the receptor by both kinases appears to be required for the full desensitization effect seen with the high concentration of agonist, which includes both losses in sensitivity and in the maximal responsiveness of the adenylyl cyclase response upon subsequent challenge with the agonist. Such a dual kinase control of BAR phosphorylation may have important implications for understanding the regulation of desensitization under different physiological circumstances.

Chemical modification of amino acid residues was used to probe the ligand recognition site of A1 adenosine receptors from rat brain membranes. The effect of treatment with group-specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N6-[3H] phenylisopropyladenosine ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). Antagonists protected A1 receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX binding from inactivation, it is concluded that agonists and antagonists occupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of the inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [3H]PIA and [3H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the ligand binding site. In addition, it was shown that no further amino acids with polar side chains are present. The absence of hydrophilic amino acids from the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A1 receptors.

The binding of agonists and antagonists to Ri adenosine receptors of synaptosomal membranes from rat and bovine brain was studied. The effects of guanine nucleotides and temperature were analyzed with the aid of computerized curve fitting. Evidence is presented for two different states of the receptor: one of high and one of low affinity for agonists. Antagonists bind to both states with the same affinity. The two states are characterized by saturation, competition, and kinetic experiments with very similar results. Guanine nucleotides cause transition of the high- to the low-affinity state. The ratio of the KD values for the two affinity states is 90-150 in rat brain but only 10 in bovine brain. The proportions of the two affinity states are the same for all agonists tested; in the absence of exogenous guanine nucleotides, 75% of the total receptor population is in the high-affinity state, whereas in the presence of guanine nucleotides only 5% remain in the high-affinity state. Binding of antagonists to the receptor is enthalpy-driven whereas binding of the agonist (-)-N6-phenylisopropyladenosine to the high-affinity state of the receptor is entropy-driven. Binding of the agonist to the low-affinity state is enthalpy-driven and thus similar to the binding of antagonists. Our data indicate that guanine nucleotides convert the Ri adenosine receptor from a high- to a low-agonist affinity state and that agonist binding shows thermodynamic differences from antagonist binding only when it is to the high-affinity state of the receptor.

The binding of agonists and antagonists to alpha 2-adrenergic receptors of human platelets was studied. The receptors showed homogeneous affinities for antagonists but two affinity states for the agonist (-)-epinephrine, which were modulated by guanine nucleotides. Van't Hoff plots of antagonist binding had a break point at about 18 degrees and considerable diversity between 18 degrees and 0 degree. Agonist binding to both affinity states showed a similar break point; agonist binding to the high affinity state was characterized by a large entropy component compared to the low affinity state. This entropy component was reduced at higher concentrations of sodium, indicating that it may be due to liberation of sodium ions. Measurements of the fluorescence of 1-anilin-8-naphthalenesulfonate showed thermotropic phase transitions of the platelet membranes at about 17 degrees. The transition temperature was decreased to about 12 degrees by addition of 10 mM octanoic acid. Octanoic acid also shifted the break points of the van't Hoff plot of antagonist and low affinity agonist binding from 18 degrees to 12 degrees. High affinity agonist binding, however, remained unchanged. It is concluded that agonist-specific thermodynamic characteristics of ligand binding to alpha 2-receptors of human platelets can only be investigated by regarding differences between high and low affinity agonist binding. These differences include an entropy increase upon ligand binding, which is in part due to enhanced liberation of sodium ions, and a loss of sensitivity to fluidity changes in the outer layer of the plasma membrane.

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor.

Exposure of beta-adrenergic receptors (beta ARs) to agonists causes rapid desensitization of the receptor-stimulated adenylyl cyclase response. Three main mechanisms have been implicated in this process: phosphorylation of the receptors by the cAMP-dependent protein kinase (PKA), phosphorylation by the specific agonist-dependent beta AR kinase, and sequestration of the receptors away from the cell surface. By applying inhibitors of these processes to digitonin-permeabilized A431 cells we investigated their contributions to beta AR desensitization. Each process could be selectively inhibited: PKA-dependent phosphorylation by an inhibitor peptide (amino acids 1-24 of the heat-stable inhibitor of PKA (PKI], beta AR kinase-dependent phosphorylation by heparin, and sequestration by concanavalin A. In permeabilized cells, heparin plus PKI completely blocked agonist-induced phosphorylation of the beta ARs. Desensitization was assessed by quantitating the signal transduction efficacy of the system. At high agonist concentrations (approximately 1 microM) up to 70% desensitization occurred. Complete blockade of this desensitization required the concurrent inhibition of all three pathways. When individual pathways were blocked it could be demonstrated that either the PKA or beta AR kinase mechanisms alone resulted in 40-50% desensitization whereas sequestration alone caused 20-30% desensitization. At low agonist concentrations (approximately 10 nM), the PKA pathway was selectively activated. These data indicate that while desensitization mediated via the three different mechanisms can occur independently, the quantitative contributions are not additive. Such findings suggest distinct but overlapping physiological roles for each mechanism in controlling receptor function.

The positive inotropic effect of the alpha-1 adrenoceptor agonist phenylephrine was accompanied by a concentration-dependent increase in inositol trisphosphate (IP3) in electrically driven left auricles isolated from rat hearts. Further analysis of the myocardial phosphoinositide pathway revealed an additional increase in inositol phosphate and inositol bisphosphate with a concomitant decrease in phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. The decrease in phosphatidylinositol bisphosphate and increase in IP3 preceded the increase in force of contraction. All effects were antagonized by the alpha-1 adrenoceptor antagonist prazosin. For comparison the effects of the beta adrenoceptor agonist isoprenaline were studied. Isoprenaline produced a positive inotropic effect similar to that of phenylephrine but all phosphoinositide products remained unaffected. The influence of lithium and calcium ions were studied systematically. The stimulatory effect of phenylephrine on inositol phosphates was lithium-dependent. Without lithium phenylephrine did not detectably affect the phosphoinositide turnover. Phenylephrine caused a maximal increase in inositol phosphate, inositol bisphospate and IP3 at 10 mmol/l of lithium. Lithium itself had a concentration-dependent positive inotropic effect. However, lithium did not enhance the positive inotropic effect of phenylephrine. Variation of the extracellular concentration of calcium did not influence the stimulatory effect of phenylephrine on inositol phosphates indicating that inositol phosphate turnover does not require the presence of extracellular calcium. It is concluded that the stimulation of myocardial phosphoinositide breakdown generating an increased IP3 turnover may be involved in the mechanism(s) whereby alpha-1 adrenoceptor stimulation exerts an increase in myocardial force of contraction.