These results highlight differential subcellular localization for betaAR subtypes and indicate that betaAR may have specific roles in regulating nuclear function in cardiomyocytes.
Angiotensin-II (Ang-II) from extracardiac sources and intracardiac synthesis regulates cardiac homeostasis, with mitogenic and growth-promoting effects largely due to altered gene expression. Here, we assessed the possibility that angiotensin-1 (AT1R) or angiotensin-2 (AT2R) receptors on the nuclear envelope mediate effects on cardiomyocyte gene expression. Immunoblots of nucleus-enriched fractions from isolated cardiomyocytes indicated the presence of AT1R and AT2R proteins that copurified with the nuclear membrane marker nucleoporin-62 and histone-3, but not markers of plasma (calpactin-I), Golgi (GRP-78), or endoplasmic reticulum (GM130) membranes. Confocal microscopy revealed AT1R and AT2R proteins on nuclear membranes. Microinjected Ang-II preferentially bound to nuclear sites of isolated cardiomyocytes. AT1R and AT2R ligands enhanced de novo RNA synthesis in isolated cardiomyocyte nuclei incubated with [␣-
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...
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