The endothelins are a remarkable family of signaling peptides: molecular biology predicted the existence of their receptors and synthetic enzymes prior to both the identification of the encoded proteins and the synthesis of antagonists and inhibitors for use as pharmacological tools. Although considerable advances have been made, culminating in the design of endothelin antagonists with the- peutic potential in cardiovascular disease, much remains to be discovered. Tantalizingly, new research frontiers are emerging. To support further progress, Peptide Research Protocols: Endothelin encompasses experimental protocols that interrogate all facets of an endogenous mammalian peptide s- tem, from peptide and receptor expression through synthetic pathway to peptide function and potential role in human disease. Chapters describe the use of molecular techniques to quantify the expression of mRNA for both endothelin receptors and the endothelin-converting enzymes. Peptides, precursors, receptors, and synthetic enzymes may be localized and quantified in plasma, culture supernatants, tissue homogenates, and tissue s- tions using antibodies, while additional information on receptor characterization may be obtained using radioligand binding techniques. Several protocols cover in vitro assays that determine the function of the endothelin peptides in isolated preparations, that characterize new endothelin receptor ligands, or provide inf- mation on the tissue-specific processing of endothelin precursor peptides.

A comprehensive collection of readily reproducible methods for studying receptors in silico, in vitro, and in vivo. These cutting-edge techniques cover mining from curated databases, identifying novel receptors by high throughput screening, molecular methods to identify mRNA encoding receptors, radioligand binding assays and their analysis, quantitative autoradiography, and imaging receptors by positron emission tomography (PET). Highlights include phenotypic characterization of receptors in knockout mice, imaging receptors using green fluorescent protein and fluorescent resonance energy transfer, and quantitative analysis of receptor mRNA by TaqMan PCR. These book equips the researcher with techniques for exploring the unprecedented number of new receptor systems now emerging and the so-called "orphan" receptors whose activating ligand has not been identified.

A broad definition of a receptor is a specialized protein on or in a cell that recognizes and binds a specific ligand to undergo a conformational change, leading to a physiological response or change in cell function. A ligand can be an endogenous neurotransmitter, hormone, paracrine/autocrine factor, or a synthetic drug that may function as an agonist or antagonist. The third edition of Receptor Binding Techniques expands upon the methods and techniques used for studying receptors in silico, in vitro and in vivo. Comprehensive chapters describe how to use online resources for experimental research such as prediction of receptor-ligand interactions and mine the IUPHAR receptor database. Classical techniques of radioligand binding, quantitative autoradiography and their analyses are complemented by the use of immunocytochemistry for the cellular localization of receptor protein and hybridization to detect receptor mRNA. Protocols using fluorescent labeled ligands are described to visualise receptors in living cells, their interaction with beta-arrestin to measure ligand-induced internalisation and green fluorescent protein to study trafficking. Non-radioactive, chemiluminescent cAMP and arrestin assays facilitate the identification of novel 'biased agonists'. Detailed methods are provided for in vivo imaging of receptors using positron emission tomography (PET). Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Receptor Binding Techniques, Third Edition, aids scientists in continuing to study receptor binding.

A broad definition of a receptor is a specialized protein on or in a cell that recognizes and binds a specific ligand to undergo a conformational change, leading to a physiological response or change in cell function. A ligand can be an endogenous neurotransmitter, hormone, paracrine/autocrine factor, or a synthetic drug that may function as an agonist or antagonist. The third edition of Receptor Binding Techniques expands upon the methods and techniques used for studying receptors in silico, in vitro and in vivo. Comprehensive chapters describe how to use online resources for experimental research such as prediction of receptor-ligand interactions and mine the IUPHAR receptor database. Classical techniques of radioligand binding, quantitative autoradiography and their analyses are complemented by the use of immunocytochemistry for the cellular localization of receptor protein and hybridization to detect receptor mRNA. Protocols using fluorescent labeled ligands are described to visualise receptors in living cells, their interaction with beta-arrestin to measure ligand-induced internalisation and green fluorescent protein to study trafficking. Non-radioactive, chemiluminescent cAMP and arrestin assays facilitate the identification of novel 'biased agonists'. Detailed methods are provided for in vivo imaging of receptors using positron emission tomography (PET). Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Receptor Binding Techniques, Third Edition, aids scientists in continuing to study receptor binding.

The endothelins are a remarkable family of signaling peptides: molecular biology predicted the existence of their receptors and synthetic enzymes prior to both the identification of the encoded proteins and the synthesis of antagonists and inhibitors for use as pharmacological tools. Although considerable advances have been made, culminating in the design of endothelin antagonists with the- peutic potential in cardiovascular disease, much remains to be discovered. Tantalizingly, new research frontiers are emerging. To support further progress, Peptide Research Protocols: Endothelin encompasses experimental protocols that interrogate all facets of an endogenous mammalian peptide s- tem, from peptide and receptor expression through synthetic pathway to peptide function and potential role in human disease. Chapters describe the use of molecular techniques to quantify the expression of mRNA for both endothelin receptors and the endothelin-converting enzymes. Peptides, precursors, receptors, and synthetic enzymes may be localized and quantified in plasma, culture supernatants, tissue homogenates, and tissue s- tions using antibodies, while additional information on receptor characterization may be obtained using radioligand binding techniques. Several protocols cover in vitro assays that determine the function of the endothelin peptides in isolated preparations, that characterize new endothelin receptor ligands, or provide inf- mation on the tissue-specific processing of endothelin precursor peptides.

A comprehensive collection of readily reproducible methods for studying receptors in silico, in vitro, and in vivo. These cutting-edge techniques cover mining from curated databases, identifying novel receptors by high throughput screening, molecular methods to identify mRNA encoding receptors, radioligand binding assays and their analysis, quantitative autoradiography, and imaging receptors by positron emission tomography (PET). Highlights include phenotypic characterization of receptors in knockout mice, imaging receptors using green fluorescent protein and fluorescent resonance energy transfer, and quantitative analysis of receptor mRNA by TaqMan PCR. These book equips the researcher with techniques for exploring the unprecedented number of new receptor systems now emerging and the so-called "orphan" receptors whose activating ligand has not been identified.