Localization of functional endothelin receptor signaling complexes in cardiac transverse tubules.
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 ETA 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 125I-ET-1 to provide evidence for ET-1 receptors in cardiac transverse tubules (T-tubules). Moreover, the ETA receptor and downstream effector phospholipase C-beta 1 were co-localized within T-tubules using standard immunofluorescence techniques, and protein kinase C (PKC)-epsilon-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 nM ET-1, despite retaining both a nearly normal twitch response to field stimulation and a robust positive inotropic response to 20 nm isoproterenol. We propose that ET-1 enhances myocyte contractility by activating ETA receptor-phospholipase C-beta 1-PKC-epsilon 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.
Robu
Valentin G
VG
Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Pfeiffer
Emily S
ES
Robia
Seth L
SL
Balijepalli
Ravi C
RC
Pi
YeQing
Y
Kamp
Timothy J
TJ
Walker
Jeffery W
JW
D000818:Animals; D015153:Blotting, Western; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000378:metabolism; D019793:Fluorescein / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006352:Heart Ventricles / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008956:Models, Chemical; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000502:physiology*; D054799:Phospholipase C beta; D017785:Photons; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D051744:Protein Kinase C-epsilon; D051381:Rats; D017207:Rats, Sprague-Dawley; D017466:Receptors, Endothelin / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D019332:Endothelin-1; D007527:Isoenzymes; D008164:Luminescent Proteins; D017466:Receptors, Endothelin; D049452:Green Fluorescent Proteins; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D007545:Isoproterenol; D019793:Fluorescein