Functional Endothelin Receptors Are Present on Nuclei in Cardiac Ventricular Myocytes

Abstract

Endothelins are thought to act through two specific, plasmalemmal G protein-coupled receptor subtypes, ET A R and ET B R. However, in subfractionated cardiac membranes, ET A R immunoreactivity was detected only in the plasma membrane whereas ET B R immunoreactivity was detected predominantly in membranes of intracellular origin. Confocal microscopy demonstrated the presence of intracellular ET A R and ET B R in ventricular myocytes. ET A R were primarily on plasma membrane (surface membranes and transverse-tubules) and to a lesser extent on the nucleus while ET B R localized primarily to the nuclei. Western blot analysis of nuclei isolated from the heart indicated the presence of endothelin receptors: both ET A R and ET B R copurified with nucleoporin 62, whereas markers of endoplasmic reticulum and Golgi membranes were depleted. Endothelins are a family of 21-amino acid isopeptides (ET-1, -2, and -3), 1 derived from different genes, which mediate a wide spectrum of pharmacological activities in a variety of tissues (see Ref. 1). In the heart, ET-1 produces positive inotropic (2-4) and chronotropic (5) effects, prolongs the action potential (6, 7), and mediates cardiac remodeling in hypertrophy (4, 8 -14), myocardial infarction (15), and congestive heart failure (16, 17). To date, two mammalian endothelin receptor subtypes (ET A R and ET B R) have been cloned (18 -20). The ET A R is selective for ET-1 ϭ ET-2 Ͼ Ͼ ET-3, with sarafotoxin 6c being inactive whereas the ET B R is non-selective. Subtype-specific pharmacological antagonists also help to distinguish the two receptor subtypes. An additional endothelin receptor (ETR) subtype, ET C R, has been cloned from Xenopus laevis (21); however, a mammalian homolog has yet to be identified. Both ET A R and ET B R are seven-transmembrane spanning receptors known to couple to an overlapping array of heterotrimeric G-proteins (22) leading to activation of multiple signaling systems including phospholipase C (23-25), phospholipase D (26, 27), phospholipase A 2 (28), cytosolic Ca 2ϩ (29, 30), Na/H exchange (31), cAMP production (23), cGMP production (32), tyrosine kinases (33, 34), and mitogen-activated protein kinases (14,35,36). Both ET A R and ET B R subtypes are present in heart (18,19,(37)(38)(39); in human myocardium, ET A R and ET B R are expressed at similar levels (38).It is now thought that ET-1 may act in an autocrine/paracrine manner in the cardiac ventricular myocyte. All three endothelins are synthesized as larger precursor proteins, prepro-ETs, which are subsequently cleaved to 37-41-amino acid proforms, referred to as big endothelins. Big endothelins are converted to mature endothelins by endothelin-converting enzymes (ECE). Splice variants of the ECE-1 isoform, ECE-1a and ECE-1c, have been detected in adult cardiac myocytes (40), and ECE-1c expression is up-regulated 5-fold in myocytes during congestive heart failure (40). ET-1 is produced, stored, and secreted by neonatal (41) and adult cardiac ventricular myocytes (42) under basal conditions, and regulat...

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2+ in adult cardiac myocytes

“…However, endothelin receptors, as well as several other GPCRs and their effectors, are also present on cardiac nuclear membranes (reviewed in [9][10][11]). Immunocytofluorescence studies have revealed ETA to be primarily in the plasma membrane with some perinuclear staining, whereas ETB localize to the nuclear membrane in ventricular myocytes [12,13]. The presence of GPCR effector molecules at the nuclear envelope or within the nucleus further supports the concept of GPCR signalling at the nuclear membrane [14].…”

Nuclear β-adrenergic receptors modulate gene expression in adult rat heart

“…Rat cardiac nuclei were isolated as described previously [12]. Briefly, rat hearts were pulverized under liquid nitrogen, resuspended in cold PBS, and homogenized (Polytron, 8000 rpm; 2×10 s, Fraction 1).…”

“…SDS PAGE and immunoblotting were performed as previously described [12]. Immunoreactive bands were quantified using a BioRad VersaDoc 4000 imaging system and Quantity One® software (Version 4.5.2 for Mac).…”

“…Freshly isolated nuclei were incubated with ISO (1 μM), in the absence or presence of an inhibitor of either MEK1/2 activation (PD98059) or activity (U0126), and [α 32 P]UTP to measure initiation of RNA transcription. Endothelin type B receptors (ETB) have also been shown to be present on the nuclear envelope [12]; hence, purified nuclei were also treated with endothelin 1 (ET-1, 10 nM). Interestingly, whereas ISO increased [ 32 P]UTP incorporation (Fig.…”

Emerging roles of inositol 1,4,5-trisphosphate signaling in cardiac myocytes

“…In addition, many receptors that act as signal transducers at the cell membrane are also found in the nuclear envelope (e.g. ET A and ET B receptors [28]) where they might activate a nuclear PIP 2 -PLC-IP 3 cascade. The mechanism for stimulation of nuclear hormone receptors is not entirely clear, although it has been suggested that activated receptors can be internalized from the surface of cells and translocated to the nucleus where they engage signaling pathways.…”