Full Description
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.
Contents
Endothelin Peptide Protocols.- Immunocytochemical Localization of Endothelin Peptides, Precursors, and Endothelin-Converting Enzymes.- Analysis of Endothelins by Enzyme-Linked Immunosorbent Assay and Radioimmunoassay.- Separation of Endothelin Isoforms by High Performance Liquid Chromatography and Detection by Radioimmunoassay.- Endothelin Receptor Protocols.- Radioligand binding assays and quantitative autoradiography of endothelin receptors.- Using Receptor Antagonists in Binding Studies to Characterize a Mammalian Endothelin Receptor.- Quantification of Endothelin Receptor mRNA by Competitive RT-PCR.- Endothelin-Converting Enzyme Protocols.- Detection and Assessment of Endothelin-Converting Enzyme Activity.- Quantitative Measurement of mRNA Levels by RT-PCR.- Evaluation of Endothelin-Converting Enzyme Inhibitors Using Cultured Cells.- Endothelin-Converting Enzyme Activity in Vascular Smooth Muscle Preparations In Vitro.- In Vitro Functional Assays.- Characterization of a New Endothelin Receptor Ligand by In Vitro Assays.- Use of In Vitro Organ Cultures of Human Saphenous Vein as a Model for Intimal Proliferation.- In Vivo Functional Assays.- Investigation of the Endothelin System in Experimental Heart Failure.- Local Forearm Vasoconstriction to Endothelin-1 Measured by Venous Occlusion Plethysmography In Vivo.
