Angiotensin II AT1 receptor internalization, translocation and de novo synthesis modulate cytosolic and nuclear calcium in human vascular smooth muscle cells

• Published in: Canadian journal of physiology and pharmacology 2003-03, Vol.81 (3), p.274-287
• Main Authors: Bkaily, G, Sleiman, S, Stephan, J, Asselin, C, Choufani, S, Kamal, M, Jacques, D, Gobeil, Jr, F, D'Orléans-Juste, P
• Format: Article
• Language: English
• Subjects: Aniline Compounds; Aorta - cytology; Aorta - metabolism; Biological and medical sciences; Blood vessels and receptors; Blotting, Western; Calcium; Calcium - metabolism; Cell Nucleus - metabolism; Cells; Cells, Cultured; Fluorescent Antibody Technique; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Green Fluorescent Proteins; Humans; Luminescent Proteins - genetics; Microscopy, Confocal; Muscle, Smooth, Vascular - cytology; Muscle, Smooth, Vascular - metabolism; Muscles; Receptor, Angiotensin, Type 1 - genetics; Receptor, Angiotensin, Type 1 - metabolism; Recombinant Fusion Proteins - metabolism; Sarcolemma - physiology; Transfection; Translocation, Genetic; Veins & arteries; Vertebrates: cardiovascular system; Xanthenes
• ISSN: 0008-4212; 1205-7541
• DOI: 10.1139/y03-007

The present study was designed to verify if human (h) Angiotensin II (Ang II) type-1 receptor (hAT 1 R) undergoes internalization, nuclear translocation, and de novo synthesis in primary culture of human aortic vascular smooth muscle cells (hVSMCs) and if overexpression of this receptor modulates sustained free cytosolic ([Ca] c ) and nuclear ([Ca] n ) calcium. 3-dimensional (3-D) confocal microscopy was used to monitor free intracellular Ca 2+ and hAT 1 R-green fluorescence protein (GFP) fusion protein in cultured hVSMCs. Immunofluorescence studies showed the presence of hAT 1 R and the absence of hAT 2 R in normal hVSMCs. Using 3-D imaging technique, hAT 1 receptors were localized at the sarcolemma and in the cytosolic and nuclear compartments. In native as well as in normal hAT 1 R or hAT 1 R –GFP overexpressing hVSMCs, Ang II (10 –9 and 10 –4 M) induced internalization and nuclear translocation of this type of receptor. The internalization of hAT 1 Rs is mediated via clathrin-coated pits and vesicles pathway. This phenomenon of trancellular trafficking of receptors was associated with an increase of hAT 1 R. The Ang II induced increase of hAT 1 R density was prevented by the protein synthesis inhibitor cycloheximide. Overexpression of hAT 1 R and hAT 1 R–GFP decreased both basal cytosolic and nuclear Ca 2+ . In normal hVSMCs and low hAT 1 R–GFP overexpressing hVSMCs, Ang II (10 –15 to 10 –4 M) induced a dose-dependent sustained increase of [Ca] c and [Ca] n with an EC 50 near 5 × 10 –11 and 5 × 10 –9 M, respectively. Our results suggest that hAT 1 Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and the nuclear compartments. Ang II rapidly induces hAT 1 R internalization, nuclear translocation, as well as nuclear de novo synthesis of this receptor. The hAT 1 R overexpression in hVSMCs modulates sustained [Ca] c and [Ca] n .Key words: angiotensin, calcium, protein synthesis, nucleus, AT 1 receptor, nuclear de novo synthesis.