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

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 su...

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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
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Language:English
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Summary: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.
ISSN:0008-4212
1205-7541
DOI:10.1139/y03-007