Using Caged Ligands to Study Intracrine Endothelin Signaling in Intact Cardiac Myocytes

Abstract

Intracrine signaling refers to the activation of receptors located within the cell and many intracrine receptors have been localized to the nuclear membrane. The presence and function of nuclear receptors have been studied in isolated nuclei. Much less information is available concerning the function of these receptors within the context of intact cells due, in part, to difficulties in accessing the intracellular receptor without activating those at the cell surface. Here, we describe the use of caged agonists to study intracrine signaling in intact, living cells. The caging moiety permits cells to be loaded with a functionally “inert” ligand. After washing the cells free of extracellular caged ligand, a brief exposure to UV releases the native ligand within the cell. The actual duration of UV irradiation required is a function of the type of caging moiety employed and where it is incorporated into the ligand. Cells may then be assessed for changes in morphology, second messenger production, cellular signaling, or gene expression by confocal fluorescence microscopy, immunoblotting, or transcriptomic techniques.