Canine Mast Cell Adenosine Receptors: Cloning and Expression of the A3 Receptor and Evidence that Degranulation Is Mediated by the A2B Receptor
01 Nov 1997-Molecular Pharmacology (American Society for Pharmacology and Experimental Therapeutics)-Vol. 52, Iss: 5, pp 846-860
TL;DR: The results suggest that the cloned canine A3 AR is structurally and pharmacologically more similar to human than to rat A3AR, and although A2B receptors play a major role in the regulation of BR mast cell degranulation, multiple AR subtypes and G proteins may influence mast cell functions.
Abstract: We cloned and characterized the canine A3 adenosine receptor (AR) and examined AR-induced degranulation of the BR line of canine mastocytoma cells. Canine A3AR transcript is found predominantly in spleen, lung, liver, and testes and encodes a 314-amino acid heptahelical receptor.125I-N6-Aminobenzyladenosine binds to two affinity states of canine A3AR withKD values of 0.7 ± 0.1 and 16 ± 0.8 nm, reflecting G protein-coupled and -uncoupled receptors, respectively. Xanthine antagonists bind with similar affinities to human, canine, and rabbit receptors but with 80–400-fold lower affinities to rat A3AR. Although canine BR mastocytoma cells contain A1AR, A2BAR, and A3AR, degranulation seems to be mediated primarily by A2BARs stimulated by the nonselective agonist 5′-N-ethylcarboxamidoadenosine (NECA) but not by the A3-selective agonistN6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide. NECA-stimulated degranulation is not prevented by pertussis toxin and is blocked by enprofylline (Ki = 7 μm), an antiasthmatic xanthine with low affinity (Ki > 100 μm) for A1AR, A2AAR, and A3AR. NECA increases canine mastocytoma cell cAMP, Ca2+, and inositol trisphosphate levels; these responses are antagonized half-maximally by 7–15 μm enprofylline. The results suggest that (i) the cloned canine A3AR is structurally and pharmacologically more similar to human than to rat A3AR; (ii) the A2BAR, and not the A1AR or A3AR, is principally responsible for adenosine-mediated degranulation of canine BR mastocytoma cells; and (iii) the BR cell A2BAR couples to both Ca2+ mobilization and cAMP accumulation. Although A2B receptors play a major role in the regulation of BR mast cell degranulation, multiple AR subtypes and G proteins may influence mast cell functions.
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TL;DR: In this review particular emphasis is placed on the discrepancy between the concentrations ofadenosine, ADP, and ATP in the purine receptors of UDP and UTP.
Abstract: ### A. Overview
Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the
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Journal Article•
TL;DR: Experiments with receptor antagonists and mice with targeted disruption of adenosine A(1), A(2A), and A(3) expression reveal roles for these receptors under physiological and particularly pathophysiological conditions.
Abstract: Four adenosine receptors have been cloned and characterized from several mammalian species. The receptors are named adenosine A(1), A(2A), A(2B), and A(3). The A(2A) and A(2B) receptors preferably interact with members of the G(s) family of G proteins and the A(1) and A(3) receptors with G(i/o) proteins. However, other G protein interactions have also been described. Adenosine is the preferred endogenous agonist at all these receptors, but inosine can also activate the A(3) receptor. The levels of adenosine seen under basal conditions are sufficient to cause some activation of all the receptors, at least where they are abundantly expressed. Adenosine levels during, e.g., ischemia can activate all receptors even when expressed in low abundance. Accordingly, experiments with receptor antagonists and mice with targeted disruption of adenosine A(1), A(2A), and A(3) expression reveal roles for these receptors under physiological and particularly pathophysiological conditions. There are pharmacological tools that can be used to classify A(1), A(2A), and A(3) receptors but few drugs that interact selectively with A(2B) receptors. Testable models of the interaction of these drugs with their receptors have been generated by site-directed mutagenesis and homology-based modelling. Both agonists and antagonists are being developed as potential drugs.
2,582 citations
Cites background from "Canine Mast Cell Adenosine Receptor..."
...Furthermore, endogenous A2B receptors of HEK 293 cells, human HMC-1 mast cells and canine BR mast cells are dually coupled to Gs and Gq (Auchampach et al., 1997; Linden et al., 1999)....
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...In contrast, the A2B receptor has been implicated as the receptor subtype that facilitates the release of allergic mediators from canine and human HMC-1 mastocytoma cells (Feoktistov and Biaggioni, 1995; Auchampach et al., 1997)....
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TL;DR: Recent advances in the understanding of the roles of the various adenosine receptor subtypes, and in the development of selective and potent ligands, have brought the goal of therapeutic application of adenosines receptor modulators considerably closer.
Abstract: Adenosine receptors are major targets of caffeine, the most commonly consumed drug in the world There is growing evidence that they could also be promising therapeutic targets in a wide range of conditions, including cerebral and cardiac ischaemic diseases, sleep disorders, immune and inflammatory disorders and cancer After more than three decades of medicinal chemistry research, a considerable number of selective agonists and antagonists of adenosine receptors have been discovered, and some have been clinically evaluated, although none has yet received regulatory approval However, recent advances in the understanding of the roles of the various adenosine receptor subtypes, and in the development of selective and potent ligands, as discussed in this review, have brought the goal of therapeutic application of adenosine receptor modulators considerably closer
1,303 citations
TL;DR: In the 10 years since the previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors, no developments have led to major changes in the recommendations, but there have been so many other developments that an update is needed.
Abstract: In the 10 years since our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors, no developments have led to major changes in the recommendations. However, there have been so many other developments that an update is needed. The fact that the structure of one of the adenosine receptors has recently been solved has already led to new ways of in silico screening of ligands. The evidence that adenosine receptors can form homo- and heteromultimers has accumulated, but the functional significance of such complexes remains unclear. The availability of mice with genetic modification of all the adenosine receptors has led to a clarification of the functional roles of adenosine, and to excellent means to study the specificity of drugs. There are also interesting associations between disease and structural variants in one or more of the adenosine receptors. Several new selective agonists and antagonists have become available. They provide improved possibilities for receptor classification. There are also developments hinting at the usefulness of allosteric modulators. Many drugs targeting adenosine receptors are in clinical trials, but the established therapeutic use is still very limited.
1,145 citations
TL;DR: This work has generated excitement regarding the potential use of adenosine-receptor-based therapies in the treatment of infection, autoimmunity, ischaemia and degenerative diseases.
Abstract: Adenosine is a key endogenous molecule that regulates tissue function by activating four G-protein-coupled adenosine receptors: A1, A2A, A2B and A3. Cells of the immune system express these receptors and are responsive to the modulatory effects of adenosine in an inflammatory environment. Animal models of asthma, ischaemia, arthritis, sepsis, inflammatory bowel disease and wound healing have helped to elucidate the regulatory roles of the various adenosine receptors in dictating the development and progression of disease. This recent heightened awareness of the role of adenosine in the control of immune and inflammatory systems has generated excitement regarding the potential use of adenosine-receptor-based therapies in the treatment of infection, autoimmunity, ischaemia and degenerative diseases.
1,072 citations
References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
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Additional excerpts
...Marquardt’s nonlinear least-squares interpolation (18)....
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TL;DR: The analysis described shows K I does not equal I 50 when competitive inhibition kinetics apply; however, K I is equal to I 50 under conditions of either noncompetitive or uncompetitive kinetics.
12,583 citations
"Canine Mast Cell Adenosine Receptor..." refers methods in this paper
...Failure to analytically resolve the two agonist affinity states in radioligand binding assays will result in underestimation of high affinity agonist dissociation constants as well as errors in the calculation of the dissociation constants of competing compounds based on the Cheng and Prusoff formula (30)....
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Book•
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01 Jan 1989
TL;DR: To develop a program to print the barcodes using two commonly uses command sets and hence evaluates their ease of use for such applications, students should be able to program dot matrix printers, by manipulating bit level information and ink jet printers using page description language, such as PCL.
Abstract: 1. OBJECTIVE To develop a program to print the barcodes using two commonly uses command sets and hence evaluates their ease of use for such applications. Upon completion of this experiment, students should be able to program dot matrix printers, by manipulating bit level information and ink jet printers using page description language, such as PCL which uses raster image for graphics output. Students will also become familiar with the encoding and dimensional requirements of barcode symbologies, and the suitability of the selected printers for barcode printing. The Computer Engineering Laboratory is set up with IBM-compatible PC's installed with standard C compiler. The laboratory also has several kinds of printers for this experiment. They are classified into two groups. One group consists of dot-matrix printers and the other group consists of HP ink jet printers. In this experiment, each student will have to program a dot-matrix printer using Epson 9-pin graphics command codes in group A printers and HP PCL graphics commands for the ink jet printer in group B. The assignment of different printers is as follows: 4. INTRODUCTION Barcode labelling is widely implemented in the retail marketplace and is gaining increasing visibility in a broad range of non-retail applications. To read the information contained in a barcode symbol, a scanning device such as a wand can be used. As the scanning device is moved across the symbol, the width pattern of the bars and the spaces is analysed by the reading equipment and the original data is recovered. A number of barcodes has been developed over the years, these include UPS, interleaved 2 of 5, rationalised codabar, code 39, code 128, code 93 and code 49. The American format for article numbering is the Universal Product Code (UPC). The UPC has been successfully employed in the supermarket industry since 1973. It is designed to uniquely identify a product and its manufacturer. Refer to Appendix A for UPC specifications which are necessary for this experiment. Barcode symbols can be printed with various printing technologies and on a variety of substrate labels, tags and papers. In this experiment, plain paper will be used. Hence, the quality of printed barcode will only depend on the printing mechanism. In this experiment, students will learn to program dot matrix printers by manipulating bit level information. A brief summary of the commonly used commands is attached in Appendix B and abstract from the manuals …
8,170 citations
"Canine Mast Cell Adenosine Receptor..." refers methods in this paper
...the DEAE-dextran method (16) or stably expressed in HEK 293 cells after transfection by the Ca(21) phosphate precipitation method (17)...
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TL;DR: This article reviews the use of nonlinear regression in a practical and nonmathematical manner to demystify non linear regression so that both its power and its limitations will be appreciated.
Abstract: Many types of data are best analyzed by fitting a curve using nonlinear regression, and computer programs that perform these calculations are readily available. Like every scientific technique, however, a nonlinear regression program can produce misleading results when used inappropriately. This article reviews the use of nonlinear regression in a practical and nonmathematical manner to answer the following questions: Why is nonlinear regression superior to linear regression of transformed data? How does nonlinear regression differ from polynomial regression and cubic spline? How do nonlinear regression programs work? What choices must an investigator make before performing nonlinear regression? What do the final results mean? How can two sets of data or two fits to one set of data be compared? What problems can cause the results to be wrong? This review is designed to demystify nonlinear regression so that both its power and its limitations will be appreciated.
1,270 citations