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+ signalling. 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 * Abbreviations: The abbreviations used are: 2-APB, 2-aminoethoxydiphenyl borate; A, Angiotensin II; αAR, α-adrenergic receptor;[Ca 2+ ] n , nucleoplasmic free calcium concentration; CaMKII, Ca 2+ /calmodulin-dependent protein kinase II; DMNB, 4,5-dimethoxynitrobenzyl group; DNA, deoxyribonucleic acid; DCC, 1,3-dicyclohexylcarbodiimide; DCM, dichloromethane; DTT, dithiothreitol; ECE1-a, endothelin converting enzyme 1a; cET-1, caged ET-1; caged ET-1, [Trp-ODMNB 21 ]ET-1; ET-1, endothelin 1; ET-2, endothelin 2; ET-3, endothelin 3; ETA, endothelin type A receptor; ETB, endothelin type B receptor; ETR, endothelin receptor; GPCR, G protein-coupled receptor; HDAC5; histone deacetylase 5; IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; ISO, isoproterenol; MEF2, myocyte enhancing factor-2; PBS, phosphate buffered saline; PMSF, phenylmethanesulphonylfluoride; PNGase F, peptide N-glycosidase F; qPCR, quantitative real-time polymerase chain reaction; RNA, ribonucleic acid; RyR, ryanodine receptor; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; TX-100, Triton X-100. Address correspondence to: Bruce G. Allen, Montreal Heart Institute, 5000 Belanger St,. Montréal, Québec, Canada, H1T 1C8., Telephone: (514) ] 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 traffic 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 regulate nuclear Ca 2+ signalling.

Endothelin receptor B, a candidate gene from human studies at high altitude, improves cardiac tolerance to hypoxia in genetically engineered heterozygote mice

“…The protein encoded by EDNRB is a G protein-coupled receptor that activates a phosphatidylinositol/calcium second-messenger system (26). Whereas EDNRA is present on the cell membrane, EDNRB is also present on the nuclear membrane and is associated with regulation of nuclear Ca 2+ signaling (27). The two receptors usually have opposing actions, EDNRA in vasoconstriction and EDNRB in vasodilatation.…”

Caged ligands to study the role of intracellular GPCRs

“…To and in the absence or presence of cAng-II, as described previously [16]. Cardiomyocytes were washed three times, stained with a live-cell-permeant DNA dye (DRAQ5; 1 µM) and used for Ca 2+ imaging within 1 h. Images were obtained using a Zeiss LSM 7 Duo microscope (combined LSM710 and Zeiss Live systems) with a 63x/1.4 oil Plan-Apochromat objective.…”

“…To obtain a degree of spatial and temporal resolution, or when using ligands with poor cell permeability, biologically active molecules can be pressure-injected [11] or modified by the incorporation of a "caging" moiety [12]: an easily removable functional group that both increases cell permeability and reduces affinity of the ligand for its cognate binding site. This approach has been used to study the actions of a range of molecules, including ATP [13], GTP [14], endothelin-1, [15,16], isoproterenol [17,18], phenylephrine [19], γ-aminobutyric acid [20], urotensin II [21], endothelin receptor antagonists [16], IP 3 [22,23], cofilin [24], DNA [25] and RNA [26,27] into in HEK 293 cells using polyethylenimine (PEI) at a final concentration of 9 µg/ml [29].…”

Intranuclear Signaling Cascades Triggered by Nuclear GPCRs

“…Furthermore, in human endocardial endothelial cells, in human aortic vascular smooth muscle cells and in adult rat heart, endothelin-1 (ET-1) induces an increase in nuclear Reactive Oxygen Species [30] and NO (Table 1) [25]. In cardiac nuclear membranes, the endothelin receptor is ETBR and the absence of N-glycosylation indicates that this receptor did not originate at the cell surface but traffics directly to the nuclear membrane after biosynthesis [31]. ETBR associated with the nuclear membranes regulates nuclear Ca ++ signaling through an IP3-dependent pathway and exogenous endothelins are not ligands for for this receptor on nuclear membranes [31].…”

“…In cardiac nuclear membranes, the endothelin receptor is ETBR and the absence of N-glycosylation indicates that this receptor did not originate at the cell surface but traffics directly to the nuclear membrane after biosynthesis [31]. ETBR associated with the nuclear membranes regulates nuclear Ca ++ signaling through an IP3-dependent pathway and exogenous endothelins are not ligands for for this receptor on nuclear membranes [31]. In fetal human left ventricular endocardial endothelial cells (EECLs), both plasma membrane ETAR and ETBR mediate ET-1-induced increase of intracellular calcium; this effect is mediated only by ETAR in right EECs (EECRs).…”