title: Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca²⁺ in adult cardiac myocytes.
abstract: Endothelin receptors are present on the nuclear membranes in adult cardiac ventricular myocytes. The objectives of the present study were to determine 1) which endothelin receptor subtype is in cardiac nuclear membranes, 2) if the receptor and ligand traffic from the cell surface to the nucleus, and 3) the effect of increased intracellular ET-1 on nuclear Ca(2+) signaling. Confocal microscopy using fluorescently-labeled endothelin analogs confirmed the presence of ETB at the nuclear membrane of rat cardiomyocytes in skinned-cells and isolated nuclei. Furthermore, in both cardiac myocytes and aortic endothelial cells, endocytosed ET:ETB complexes translocated to lysosomes and not the nuclear envelope. Although ETA and ETB can form heterodimers, the presence or absence of ETA did not alter ETB trafficking. Treatment of isolated nuclei with peptide: N-glycosidase F did not alter the electrophoretic mobility of ETB. The absence of N-glycosylation further indicates that these receptors did not originate at the cell surface. Intracellular photolysis of a caged ET-1 analog ([Trp-ODMNB(21)]ET-1) evoked an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n) that was attenuated by inositol 1,4,5-trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and prevented by pre-treatment with ryanodine. A caged cell-permeable analog of the ETB-selective antagonist IRL-2500 blocked the ability of intracellular cET-1 to increase [Ca(2+)]n whereas extracellular application of ETA and ETB receptor antagonists did not. These data suggest that 1) the endothelin receptor in the cardiac nuclear membranes is ETB, 2) ETB traffics directly to the nuclear membrane after biosynthesis, 3) exogenous endothelins are not ligands for ETB on nuclear membranes, and 4) ETB associated with the nuclear membranes regulates nuclear Ca(2+) signaling.
authors.affiliation: Montreal Heart Institute, Montréal, Quebec H1T 1C8, Canada.
authors.forename: Clémence
authors.initials: C
authors.lastname: Merlen
authors.forename: Nada
authors.initials: N
authors.lastname: Farhat
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Luo
authors.forename: David
authors.initials: D
authors.lastname: Chatenet
authors.forename: Artavazd
authors.initials: A
authors.lastname: Tadevosyan
authors.forename: Louis R
authors.initials: LR
authors.lastname: Villeneuve
authors.forename: Marc-Antoine
authors.initials: MA
authors.lastname: Gillis
authors.forename: Stanley
authors.initials: S
authors.lastname: Nattel
authors.forename: Eric
authors.initials: E
authors.lastname: Thorin
authors.forename: Alain
authors.initials: A
authors.lastname: Fournier
authors.forename: Bruce G
authors.initials: BG
authors.lastname: Allen
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D016232:Endothelins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D047468:Immunoprecipitation; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism; D051381:Rats; D017466:Receptors, Endothelin / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology
chemical_list: D016232:Endothelins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D017466:Receptors, Endothelin; D012433:Ryanodine; D002118:Calcium
keywords: 1,3-dicyclohexylcarbodiimide; 2-APB; 2-aminoethoxydiphenyl borate; 4,5-dimethoxynitrobenzyl group; Ang II; Ca(2+)/calmodulin-dependent protein kinase II; CaMKII; DCC; DCM; DMNB; DNA; DTT; ECE1-a; ET-1; ET-2; ET-3; ETA; ETB; ETR; Endothelin receptor; G protein-coupled receptor; GPCR; HDAC5; IP3; IP3R; ISO; MEF2; Nuclear Ca(2+); Nuclear envelope; PBS; PMSF; PNGase F; RNA; Receptor trafficking; RyR; Stimulus–transcription coupling; TCA; TFA; TX-100; Triton X-100; [Ca(2+)](n); [Trp-ODMNB(21)]ET-1; angiotensin II; cET-1; caged ET-1; deoxyribonucleic acid; dichloromethane; dithiothreitol; endothelin 1; endothelin 2; endothelin 3; endothelin converting enzyme 1a; endothelin receptor; endothelin type A receptor; endothelin type B receptor; histone deacetylase 5; inositol 1,4,5-trisphosphate; inositol 1,4,5-trisphosphate receptor; isoproterenol; myocyte enhancing factor-2; nucleoplasmic free calcium concentration; peptide N-glycosidase F; phenylmethanesulfonyl fluoride; phosphate buffered saline; qPCR; quantitative real-time polymerase chain reaction; ribonucleic acid; ryanodine receptor; trichloroacetic acid; trifluoroacetic acid; α-AR; α-adrenergic receptor