Localization of Functional Endothelin Receptor Signaling Complexes in Cardiac Transverse Tubules

• Published in: The Journal of biological chemistry Vol. 278; no. 48; pp. 48154 - 48161
• Main Authors: Robu, Valentin G, Pfeiffer, Emily S, Robia, Seth L, Balijepalli, Ravi C, Pi, YeQing, Kamp, Timothy J, Walker, Jeffery W
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 28.11.2003
• Subjects: Animals; Blotting, Western; Dogs; Dose-Response Relationship, Drug; Endothelin-1 - metabolism; Fluorescein - pharmacology; Green Fluorescent Proteins; Heart Ventricles - metabolism; Isoenzymes - metabolism; Isoproterenol - pharmacology; Kinetics; Luminescent Proteins - metabolism; Male; Microscopy, Fluorescence; Models, Biological; Models, Chemical; Myocardium - cytology; Myocardium - metabolism; Myocytes, Cardiac - metabolism; Myocytes, Cardiac - physiology; Phospholipase C beta; Photons; Protein Binding; Protein Kinase C - metabolism; Protein Kinase C-epsilon; Rats; Rats, Sprague-Dawley; Receptors, Endothelin - metabolism; Signal Transduction; Type C Phospholipases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M304396200

Endothelin-1 (ET-1) is an autocrine factor in the mammalian heart important in enhancing cardiac performance, protecting against myocardial ischemia, and initiating the development of cardiac hypertrophy. The ET A receptor is a seven-transmembrane G-protein-coupled receptor whose precise subcellular localization in cardiac muscle is unknown. Here we used fluorescein ET-1 and 125 I-ET-1 to provide evidence for ET-1 receptors in cardiac transverse tubules (T-tubules). Moreover, the ET A receptor and downstream effector phospholipase C-β 1 were co-localized within T-tubules using standard immunofluorescence techniques, and protein kinase C (PKC)-ϵ-enhanced green fluorescent protein bound reversibly to T-tubules upon activation. Localized photorelease of diacylglycerol further suggested compartmentation of PKC signaling, with release at the myocyte “surface” mimicking the negative inotropic effects of bath-applied PKC activators and “deep” release mimicking the positive inotropic effect of ET-1. The functional significance of T-tubular ET-1 receptors was further tested by rendering the T-tubule lumen inaccessible to bath-applied ET-1. Such “detubulated” cardiac myocytes showed no positive inotropic response to 20 n m ET-1, despite retaining both a nearly normal twitch response to field stimulation and a robust positive inotropic response to 20 n m isoproterenol. We propose that ET-1 enhances myocyte contractility by activating ET A receptor-phospholipase C-β 1 -PKC-ϵ signaling complexes preferentially localized in cardiac T-tubules. Compartmentation of ET-1 signaling complexes may explain the discordant effects of ET-1 versus bath applied PKC activators and may contribute to both the specificity and diversity of the cardiac actions of ET-1.